Antibody against human prostaglandin E2 receptor EP4

ABSTRACT

It is an object of the present invention to provide an antibody that binds to a human PGE 2  receptor subtype EP4 and inhibits the function of EP4, or a functional fragment thereof. It is another object of the present invention to provide a medicament comprising the aforementioned antibody or a functional fragment thereof. Mice were immunized with the human PGE 2  receptor subtype EP4, and a monoclonal antibody that suppresses the intracellular cAMP level increase induced by EP4 was screened. In addition, the CDR sequences of the obtained monoclonal antibody were determined.

TECHNICAL FIELD

The present invention relates to a monoclonal antibody against a humanprostaglandin E₂ receptor subtype EP4.

BACKGROUND ART

Prostaglandin (PG), as well as thromboxane, is a physiologically activesubstance known as “prostanoid,” and it is a lipid having a prostanoicacid skeleton. Prostanoid such as prostaglandin is biosynthesized fromarachidonic acid that is released from a membrane phospholipid by theaction of phospholipase A2. Prostaglandin is classified into groups A toJ, based on differences in the types of an oxygen atom attached to the5-membered ring thereof and a double bond. In addition, prostaglandin isclassified into groups 1 to 3, based on the number of double bonds onthe side chain of the prostanoic acid skeleton. For instance,prostaglandin E (PGE) includes PGE₁, PGE₂ and PGE₃, which are differentfrom one another in terms of the number of double bonds on theprostanoic acid skeleton side chain.

Regarding PG, PGH₂ is generated from PGG₂ that is biosynthesized fromarachidonic acid by the action of cyclooxygenase I (COX-I) orcyclooxygenase II (COX-II), and then, PGD₂, PGE₂, PGF_(2α) and the likeare generated based on a difference in the cleavage of the bond betweenoxygen atoms. Thereafter, PGA₂, PGC₂ and the like are generated fromPGE₂. Each PG generation reaction occurs by the action of a specificenzyme, and it is considered that such enzyme has tissue specificity andgenerates PG suitable for the function of each tissue.

Among various PGs, it is considered that PGE plays various importantbiological activities and that, through the mediation of its specificreceptor, PGE is associated with regulation of the immune system, aswell as vasodilatation, a decrease in blood pressure and uterinecontraction. The PGE₂ receptor is a seven transmembraneG-protein-coupled receptor, as with other PG receptors. The PGE₂receptor is abbreviated as EP, and it was revealed that EP has 4 typesof subtypes (EP1, EP2, EP3 and EP4). Each subtype is associated withvarious phenomena in vivo. That is. EP1 is associated with an increasein intracellular Ca²⁺ concentration; EP2 and EP4 are associated with anincrease in cAMP level; and EP3 is associated with a decrease in cAMPlevel (Non Patent Literature 1). The 4 types of subtypes have highhomology to one another in terms of protein structure.

It has been reported that when a low-molecular-weight compoundantagonist having high selectivity to EP4 is administered to mice thathad been induced to have experimental autoallergic encephalomyelitis orcontact hypersensitivity, accumulation of both TH1 and TH17 cells in theregional lymph node is reduced, and the progression of the disease issuppressed (Non Patent Literature 2). It has been demonstrated that PGE₂promotes production of IL-23 in dendritic cells, as a result of anincrease in cAMP level mediated by the activation of EP4. In addition,it has also been demonstrated that, in TH17 cells, PGE₂ is involved inproliferation of the TH17 cells in coordination with IL-23. Thus, it hasbeen demonstrated that an increase in cAMP level mediated by theactivation of EP4 plays an important role for intracellular signaling inTH17 cells (Non Patent Literature 3). These reports suggest that thePGE₂ receptor antagonist, in particular, an EP4-selective antagonist beeffective for the treatment of diseases caused by immunologicalabnormality, with which TH1 or TH17 is associated, such as multiplesclerosis, rheumatoid arthritis, inflammatory bowel disease and contactdermatitis (Non Patent Literature 2).

It has been reported that many types of cancer cells overexpress COX-IIwhen compared with normal cells. Moreover, it has also been reportedthat PGE₂ acts on cancer tissues or tissues around the cancer tissuesand is involved in the progression of cancer. For example, it has beendescribed that PGE₂ is involved in infiltration of refractoryinflammatory breast cancer cells or lung cancer cells into a metastatictissue (Non Patent Literatures 4 and 5). Furthermore, it has been knownthat PGE₂ is associated with proliferation of non-small cell lung cancercells, colon cancer cells, inflammatory breast cancer cells, Blymphocytes, prostatic cancer cells and melanoma, via EP4.

PGE₂ has been known to inhibit the function of NK cells which have anaction to directly attack cancer cells. One of the mechanisms of PGE₂ toinhibit the activity of NK cells is an increase in intracellular cAMPlevel mediated by the activation of EP4 (Non Patent Literature 6). Ithas also been known that Treg cells that possibly suppress immunity tocancer are activated via EP4, and the possibility of decreasing theimmune system to cancer cells in vivo has been suggested (Non PatentLiterature 7). According to these reports, it is apparent that PGE₂ isimportant for the progression of cancer. Hence, clinical studies havebeen conducted using non-selective inhibitors to COX involvedingeneration of PGE₂. However, sufficient therapeutic results could notbe obtained due to the side effects of the inhibitors. The PGE₂ receptorantagonist, in particular, an EP4-selective antagonist directlysuppresses proliferation of cancer cells and boosts the host immunesystem to cancer. Accordingly, it is anticipated that an antibody thatselectively binds to the EP4 receptor will be effective for thetreatment of various types of cancers such as breast cancer, coloncancer, lung cancer, prostatic cancer, skin cancer and B-lymphoma.

Conventionally, non-specific COX inhibitors have been applied for therelief of pain. However, it has been known that such non-specificinhibitors cause side effects such as heartburn, indigestion, nausea,abdominal distension, diarrhea, gastralgia, peptic ulcer orgastrointestinal bleeding. In recent years, COX-II selective inhibitors(e.g. celecoxib and rofecoxib) have been developed for the purpose oftreating pain. However, it has been suggested that such COX-II selectiveinhibitors develop severe cardiovascular disorder in specific patients,and thus it has been desired to develop a drug for relieving pain basedon a different mode of action. Among PGs generated by COX, PGE₂ is knownto enhance the hypersensitivity of pain sense. It has been demonstratedby multiple animal experiments that, among PGE₂ receptors, EP4 isparticularly associated with the enhancement of the hypersensitivity ofpain sense. For example, it has been known that the expression of EP4 isenhanced in the dorsal root ganglion (GRG) in a rat model ofinflammatory pain, and that a comparatively selective EP4 antagonist(AH23848) relieves the sensitivity of pain in the aforementioned model(Non Patent Literature 8). Moreover, in an analysis using EP4 knock-outmice as well, the same results could be obtained (Non Patent Literature9). These reports suggest that a pharmaceutical product for selectivelyblocking the function of EP4 be effective for the treatment of diseasesassociated with immunological abnormality, cancer and pain, while havingfewer side effects.

As methods for selectively blocking the function of EP4, severallow-molecular-weight compound antagonists have been reported. However,none of such compound antagonists have been successful as pharmaceuticalproducts. Such low-molecular-weight compound antagonists would beimproved in terms of binding selectivity to PGE₂ receptor subtypes(EP1-4) or alleviation of binding affinity for thromboxane or otherprostanoid receptors. It is concerned that the same side effects asthose of COX inhibitors are generated unless sufficient receptorselectivity is secured.

An antibody selectively binding to the EP4 receptor is expected to havehigher selectivity than low-molecular-weight compounds. Furthermore,since an antibody drug generally has a longer half-life in blood thanlow-molecular-weight compounds, it is expected to have drug effects fora long period of time by a single administration. Such antibody drug iseffective for chronic diseases (e.g. rheumatoid arthritis, colitis,cancer, etc.).

In general, as a main action mechanism of an antibody drug directed at amembrane protein (receptor), the antibody has recognized cellsexpressing the protein, and has then removed the cells based oncomplement-dependent cytotoxicity (CDC) and antibody-dependentcell-mediated cytotoxicity (ADCC). However, since CDC or ADCC isassociated with activation of inflammatory cells such as macrophage,such antibody drug is not necessarily suitable for the treatment ofdiseases caused by immunological abnormality or pain. Accordingly, whena monoclonal antibody capable of selectively inhibiting EP4 is appliedfor the treatment of diseases caused by immunological abnormality orpain, it is desirably a functional antibody that depends on neither CDCnor ADCC. That is to say, an antibody for selectively blockingEP4-dependent intracellular signaling is desirable.

To date, Japanese Patent No. 3118460 (Patent Literature 1) discloses amethod for obtaining an antibody against EP4. However, there have notyet been any reports regarding a specific antibody that EP4-specificallysuppresses the function of EP4 at a low dose and binds to neither EP1,EP2 nor EP3. In addition, it has been known that it is difficult toobtain a functional antibody against a seven-transmembrane receptor bythe general method for obtaining a monoclonal antibody described inJapanese Patent No. 3118460.

CITATION LIST Patent Literature

-   Patent Literature 1: Japanese Patent No. 3118460

Non Patent Literature

-   Non Patent Literature 1: Sugimoto et al., J. Biol. Chem., 282,    11613-11617, 2007-   Non Patent Literature 2: Yao et al., Nat. Med., 15, 633-640, 2009-   Non Patent Literature 3: Sakata et al., J Pharmacol Sci., 112 (1):    1˜5, 2010-   Non Patent Literature 4: Robertson F M Cancer. 2010 Jun. 1; 116 (11    Suppl): 2806-14.-   Non Patent Literature 5: Martinet L. Biochem Pharmacol. 2010 Sep.    15; 80 (6): 838-45.-   Non Patent Literature 6: Sharma S D, Mol Cancer Ther. 2010 March; 9    (3): 569-80.-   Non Patent Literature 7: Sharma S et al. Cancer Res. 2005 Jun. 15;    65 (12): 5211-20-   Non Patent Literature 8: Lin C.-R. et al., J Pharmacology and    Experimental Therapeutics 2006, 319: 3 (1096-1103)-   Non Patent Literature 9: Popp L. et al., European Journal of Pain.    2009, 13: 7 (691-703)

SUMMARY OF INVENTION Technical Problem

It is an object of the present invention to provide an antibody againsta human PGE₂ receptor subtype EP4, which is useful as a therapeuticagent for diseases caused by immunological abnormality, tumor and pain,a pharmaceutical composition comprising the aforementioned anti-humanEP4 antibody, and the like.

Solution to Problem

The present inventors have attempted to produce a monoclonal antibodyagainst a human PGE₂ receptor subtype EP4. As a result, the inventorshave succeeded in obtaining an antibody that specifically binds to theextracellular domain of the subtype EP4 and suppresses the function ofEP4 (e.g. the function to increase intracellular cAMP level), therebycompleting the present invention.

Specifically, the present invention includes the following (1) to (21).

(1) An antibody that binds to the extracellular domain of a PGE₂receptor subtype EP4 and inhibits the function of EP4, or a functionalfragment thereof.

(2) The antibody according to (1) above or a functional fragmentthereof, wherein the antibody is a monoclonal antibody.

(3) The antibody according to (2) above or a functional fragmentthereof, wherein the antibody is produced from hybridomas with theinternational depositary accession Nos. FERM BP-11402 and FERM BP-11403.

(4) The antibody according to any one of (1) to (3) above or afunctional fragment thereof, wherein the function of EP4 is to increasethe intracellular cAMP level.

(5) The antibody according to any one of (1) to (3) above or afunctional fragment thereof, wherein the antibody specifically binds tothe extracellular domain of EP4, and comprises any one of the following(A), (B), or (C), with regard to the amino acid sequences of itscomplementarity determining regions 1-3 (CDR1-3):(A) the antibody hasheavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:5,heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:6.heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:7,light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:8,light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:9, andlight chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:10;(B) the antibody hasheavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:15,heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:16,heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:17,light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:18,light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:19, andlight chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:20; or(C) the antibody hasheavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:45,heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:46,heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:47,light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:48,light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:49, andlight chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:50.(6) The antibody according to any one of (1) to (5) above or afunctional fragment thereof, wherein the antibody specifically binds tothe extracellular domain of EP4, and comprises any one of the following(a), (b), or (c), with regard to the amino acid sequences of its heavychain variable region and light chain variably region:(a) the antibody has a heavy chain variable region comprising the aminoacid sequence shown in SEQ ID NO: 2, and a light chain variable regioncomprising the amino acid sequence shown in SEQ ID NO: 4;(b) the antibody has a heavy chain variable region comprising the aminoacid sequence shown in SEQ ID NO: 12, and a light chain variable regioncomprising the amino acid sequence shown in SEQ ID NO: 14; or(c) the antibody has a heavy chain variable region comprising the aminoacid sequence shown in SEQ ID NO: 43, and a light chain variable regioncomprising the amino acid sequence shown in SEQ ID NO: 44.(7) An antibody that binds to the extracellular domain of EP4 andinhibits the function of EP4, or a functional fragment thereof, whereinthe antibody binds to the same epitope, to which the antibody accordingto any one of (3) to (6) above binds.(8) The antibody according to any one of (1) to (7) above or afunctional fragment thereof, wherein the antibody is a humanizedantibody or a chimeric antibody.(9) The antibody according to any one of (1) to (7) above or afunctional fragment thereof, wherein the antibody is a human antibody.(10) The antibody according to any one of (1) to (9) above or afunctional fragment thereof, wherein the antibody is an antibodyfragment, a single-chain antibody, or a diabody.(11) A nucleic acid encoding the heavy chain variable region or lightchain variable region of the antibody according to (5) or (6) above,wherein the nucleic acid is shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 41, or SEQ ID NO: 42.(12) A vector comprising the nucleic acid according to (11) above.(13) A cell into which the vector according to (12) above is introduced.(14) A pharmaceutical composition comprising the antibody according toany one of (1) to (10) above or a functional fragment thereof.(15) The pharmaceutical composition according to (14) above, which isused for prevention or treatment of a disease that develops orprogresses due to abnormality in the function of EP4.(16) The pharmaceutical composition according to (15) above, wherein thedisease to be treated is an immunological disease.(17) The pharmaceutical composition according to (15) above, wherein thedisease to be treated is tumor.(18) The pharmaceutical composition according to (15) above, wherein thedisease to be treated is pain.(19) An antibody-immobilized carrier, wherein the anti-EP4 antibodyaccording to any one of (1) to (10) above or a functional fragmentthereof is immobilized on a carrier.(20) The antibody-immobilized carrier according to (19) above, which isused such that blood comprising EP4-expressing cells is allowed to comeinto contact with the carrier, and the EP4-expressing cells are thenremoved from the body fluid.(21) A kit for measuring the expression level of EP4 on a cell surface,which comprises the anti-EP4 antibody according to any one of (1) to(10) above.

Advantageous Effects of Invention

According to the present invention, an antibody that EP4-specificallysuppresses the function of EP4 has been provided for the first time.

According to the present invention, the EP4-related medicament of thepresent invention is able to treat or prevent EP4-related immunologicaldiseases, tumor and pain. In particular, using the antibody of thepresent invention having higher binding selectivity to a subtype EP4than low-molecular-weight compounds, therapeutic effects with fewer sideeffects can be provided.

Using the antibody-immobilized carrier of the present invention,EP4-expressing cells can be selectively removed from the blood of apatient affected with cancer, autoimmune disease or the like.

Using the kit for measuring the expression level of EP4 of the presentinvention, EP4-expressing cells can be detected in the blood of apatient affected with cancer, autoimmune disease or the like, and then,using the detected cells, the condition of the disease can be evaluated.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of flow cytometry, which analyzed the bindingof a mouse isotype control antibody, NBG016-mAb14 and NBG016-mAb21 tothe parent Flp-In-CHO cell line and the CHO cell line stably expressinghuman EP4.

FIG. 2 shows the results of flow cytometry, which analyzed the bindingof a mouse isotype control antibody and NBG016-mAb9 to the parentFlp-In-CHO cell line and the CHO cell line stably expressing human EP4.

FIG. 3 shows the results obtained by analyzing the suppressive effectsof a mouse isotype control antibody, NBG016-mAb14 and NBG016-mAb21 onPGE₂-induced increase in cAMP level.

FIG. 4 shows the results obtained by analyzing the suppressive effectsof a mouse isotype control antibody and NBG016-mAb9 on PGE₂-inducedincrease in cAMP level.

FIG. 5 shows the results of flow cytometry, which analyzed the bindingof NBG016-mAb14 to 293FT cells, into which each of human EP1-4 and mouseEP1-4 genes had been introduced.

FIG. 6 shows the results of flow cytometry, which analyzed the bindingof NBG016-mAb9 to 293FT cells, into which each of human EP1-4 and mouseEP1-4 genes had been introduced.

FIG. 7 shows the results of flow cytometry, which analyzed the bindingof a mouse isotype control antibody, NBG016-mAb14 and NBG016-mAb21 tolymphocyte subsets in human peripheral blood.

FIG. 8 shows the results of flow cytometry, which analyzed the bindingof a mouse isotype control antibody and NBG016-mAb9 to lymphocytesubsets in human peripheral blood.

FIG. 9 shows the results of immunostaining the human monocytic THP1 cellline treated with PMA, using an anti-EP4 antibody.

FIG. 10 shows the results of flow cytometry, which analyzed the bindingof a culture supernatant of antibody gene-non-introduced cells and aculture supernatant of recombinant antibody NBG016-mAb9 gene-expressingcells to the parent Flp-In-CHO cell line and the CHO cell line stablyexpressing human EP4.

FIG. 11 shows the results of flow cytometry, which analyzed the bindingof a mouse isotype control antibody, the recombinant antibodyNBG016-mAb14 and the recombinant antibody NBG016-mAb21 to the parentFlp-In-CHO cell line and CHO cell line stably expressing human EP4.

FIG. 12(A) shows the results of flow cytometry, which analyzed thebinding of a mouse isotype control antibody and the mouse IgG1 antibodyNBG016-mAb21 to the parent Flp-in-CHO cell line and the CHO cell linestably expressing human EP4. FIG. 12(B) shows the results obtained byanalyzing the suppressive effects of a mouse isotype control antibodyand the mouse IgG1 antibody NBG016-mAb21 on PGE₂-induced increase incAMP level.

FIG. 13 shows the results of flow cytometry, which analyzed the bindingof a culture supernatant of antibody gene-non-introduced cells, aculture supernatant of human chimeric antibody NBG016-mAb14gene-expressing cells, and a culture supernatant of human chimericantibody NBG016-mAb21 gene-expressing cells, to the parent Flp-In-CHOcell line and the CHO cell line stably expressing human EP4.

DESCRIPTION OF EMBODIMENTS

The present invention relates to an antibody that binds to theextracellular domain of a human PGE₁ receptor subtype EP4 and suppressesthe function of EP4, or a functional fragment thereof, and a medicamentcomprising the antibody or a functional fragment thereof.

Definition of EP4 Protein

As an EP4 protein serving as an antigen in the present invention, arecombinant protein prepared from cDNA encoding the EP4 protein or thelike can be used. Alternatively, a suitable cell that expresses EP4 onthe surface thereof may be used as an antigen. A nucleic acid sequenceencoding a human EP4 protein can be searched in published database suchas GenBank (e.g. Accession No.: NM_(—)000958). Using a probe, a primerpair for PCR amplification, or the like produced based on theaforementioned gene sequence or the like, DNA (e.g. cDNA) encoding EP4can be prepared from a suitable DNA library. Alternatively, total cDNAcan be prepared by an artificial DNA synthesis method. As an example, anamino acid sequence corresponding to human EP4 is shown in SEQ ID NO:21. Human EP4 has been known to have various types of variants such asamino acid substituted variants, as well as that shown in SEQ ID NO: 21.The term “human EP4” is used in the present invention to include theaforementioned variants, as long as it has the function of EP4.

The intracellular and extracellular domains of human EP4 are consideredto correspond to the below-mentioned portions in the amino acid sequenceshown in SEQ ID NO: 21. The left term indicates amino acid numbers, andthe right term indicates the relevant domain. It is to be noted that theboundary between individual domains may include some range (1 to aminoacid residues, preferably 1 to 3 amino acid residues, and morepreferably 1 or 2 amino acid residues).

1 to 19: N-terminal domain

44 to 54: Intracellular first loop domain

80 to 96: Extracellular first loop domain

116 to 135: Intracellular second loop domain

161 to 184: Extracellular second loop domain

212 to 267: Intracellular third loop domain

296 to 312: Extracellular third loop domain

333 to 488: C-terminal domain

Definition of Antibody or Functional Antibody

The antibody of the present invention that suppresses the function ofEP4 includes a monoclonal antibody and a polyclonal antibody. Thefunctional fragments of the present antibody include antibody fragmentssuch as Fab or F(ab′)₂, and single-chain antibodies. As far as it is apolypeptide (or a polypeptide complex) that constitutes a part of anantibody and suppresses the function of EP4, all types of polypeptidesare included in the scope of the present invention.

Definition of Functional Antibody Specific to EP4 and Evaluation MethodTherefor

Examples of the function of EP4 include an increase in intracellularcAMP level and activation of Phosphoinositide 3-kinase (PI3K). It hasbeen known that these intracellular changes regulate proliferation ofcancer cells, proliferation of T lymphocytes, and generation ofcytokines. The specific binding of the antibody of the present inventionto the extracellular domain of a human PGE₂ receptor subtype EP4 can beproved as follows. A nucleic acid sequence encoding a human EP4 proteinis inserted into an expression vector, and the vector is then introducedinto suitable host cells (e.g. mammalian cells such as CHO cells, yeastcells, insect cells, etc.). Non-destructive. EP4-expressing host cellsor EP4-non-expressing host cells, which do not comprise an insertion,deletion, substitution or the like of amino acids, are allowed to comeinto contact with the antibody of the present invention, and they areallowed to react with each other for a certain period of time. Afterexcessive antibody has been washed off, the cells are subjected toELISA, RIA or flow cytometry, so that the amount of antibody binding tothe cells is measured. If a larger amount of the antibody of the presentinvention binds to the EP4-expressing host cells than to thenon-expressing host cells, the results can show the specific binding ofthe antibody of the present invention to the extracellular domain ofEP4. Moreover, there is constructed an expression vector into which anucleic acid sequence encoding a human- or mouse-derived EP1, EP2, EP3or EP4 protein has been inserted. Then, receptor-expressing host cellsare analyzed in the same manner as described above. Thus, it can bedemonstrated that the antibody of the present invention binds to humanEP4-expressing host cells more strongly than to otherreceptor-expressing cells, and preferably that the binding of theantibody of the present invention to cells that express receptors otherthan human EP4 cannot be found.

The fact that the antibody of the present invention is an antibody thatinhibits the function of EP4 can be explained as follows. A nucleic acidsequence encoding a human EP4 protein is inserted into an expressionvector, and the vector is then introduced into suitable host cells (e.g.mammalian cells such as CHO cells, yeast cells, insect cells, etc.). Thehuman EP4-expressing host cells are allowed to come into contact withthe antibody with a concentration of 0.01 to 30 μg/mL, and then, thecells are further allowed to come into contact with PGE₂ with aconcentration of 10⁻¹² to 10⁻⁶ M. Thereafter, an increase inintracellular cAMP level is measured by a suitable method. When theantibody of the present invention is added to the cells, it is able tosuppress a PGE₂-induced increase in cAMP level in a dose-dependentmanner.

Moreover, the present antibody with a concentration of 0.01 to 10 μg/mLis allowed to come into contact with a cell line that naturallyexpresses human EP4 (e.g. human macrophage cells), and PGE₂ with aconcentration of 10⁻¹² to 10⁻⁶ M is further allowed to come into contactwith the cells. Thereafter, the generation of cytokines or chemokines byinflammatory stimulus (e.g. lipopolysaccharide (LPS)) is examined. Ithas been known that PGE₂ suppresses the generation of cytokines by LPSstimulus through the mediation of EP4 or EP2. The activity of theantibody of the present invention to inhibit the function of EP4 can beevaluated using, as an indicator, the fact that the present antibodyrecovers the suppression of cytokine generation by PGE₂, through themediation of EP4. Likewise, the activity of the present antibody toinhibit the function of EP4 can also be evaluated using, as anindicator, the effect of the present antibody to inhibit the generationof IL-23 enhanced by PGE₂ from human peripheral blood dendritic cells.

Furthermore, when a cancer cell line derived from human bladder cancer,breast cancer, cervical cancer, colorectal cancer, esophageal cancer,cancer of the head and neck, skin cancer, lung cancer, oral cancer,prostatic cancer or multiple myeloma (e.g. MDA-MB-231 cells, HCA-7cells, HT-29 cells, etc.) is allowed to come into contact with PGE₂, theproliferative activity of the cells is enhanced. After the antibody ofthe present invention has previously been allowed to come into contactwith these cells, the activity of the present antibody to inhibit thefunction of PGE₂ can be evaluated using, as an indicator, the fact thatan increase in the proliferative activity of the cells caused by PGE₂ isreduced.

The antibody of the present invention binds only to human EP4 and doesnot react with mouse EP4. Accordingly, it is difficult to evaluate byanimal tests the drug effects of the antibody of the present inventionregarding immunological abnormality or pain. On the other hand,regarding the antitumor effects of the present antibody, cells highlyexpressing EP4, which have been established from the above-mentionedhuman cancer tissues, are grafted into immunodeficient mice in an amountof 10⁶ to 10⁷ cells per mouse. Immediately after the graft of the cells,the antibody of the present invention is intraperitoneally orsubcutaneously administered to the mice at a dose of 0.1 to 0.5mg/mouse. When compared with an isotype control antibody administrationgroup, tumor formation or metastasis frequency can be significantlyreduced in the administration group, to which the antibody of thepresent invention has been administered. Thus, it can be demonstratedthat the antibody of the present invention has an antitumor effect.

Detailed Definition of the Antibody of the Present Invention

Examples of the antibody of the present invention and a functionalfragment thereof include: monoclonal antibodies produced from hybridomashaving international depositary accession numbers FERM BP-11402(NBG016-mAb14) and FERM BP-11403 (NBG016-mAb21); and monoclonalantibodies prepared by the methods described in the after-mentionedExamples.

Moreover, other examples of the antibody of the present invention and afunctional fragment thereof include: an antibody which have a heavychain variable region comprising the amino acid sequence shown in SEQ IDNO: 2 and a light chain variable region comprising the amino acidsequence shown in SEQ ID NO: 4; an antibody which have a heavy chainvariable region comprising the amino acid sequence shown in SEQ ID NO:12 and a light chain variable region comprising the amino acid sequenceshown in SEQ ID NO: 14; an antibody which have a heavy chain variableregion comprising the amino acid sequence shown in SEQ ID NO: 43 and alight chain variable region comprising the amino acid sequence shown inSEQ ID NO: 44: an antibody which have a heavy chain comprising the aminoacid sequence shown in SEQ ID NO: 23 and a light chain comprising theamino acid sequence shown in SEQ ID NO: 25; an antibody which have aheavy chain comprising the amino acid sequence shown in SEQ ID NO: 27and a light chain comprising the amino acid sequence shown in SEQ ID NO:29; an antibody which have a heavy chain comprising the amino acidsequence shown in SEQ ID NO: 56 and a light chain comprising the aminoacid sequence shown in SEQ ID NO: 57, functional fragments of theaforementioned antibodies; an antibody consisting of a heavy chainand/or a light chain having an amino acid sequence(s) comprising adeletion, substitution or addition of one or several amino acids, withrespect to the amino acid sequence(s) of a heaving chain and/or a lightchain that constitute(s) the aforementioned antibodies; and functionalfragments thereof that suppress the function of EP4.

Definition of Epitope Identical to that of the Antibody of the PresentInvention

Further, a particularly preferred example of the antibody of the presentinvention and a functional fragment thereof is an antibody having anepitope overlapped with (or identical to) the epitope of any one of themonoclonal antibodies isolated in Examples. In the present invention,such an antibody is referred to as an antibody binding to substantiallythe same site. Whether or not two antibodies bind to substantially thesame site can be determined, for example, by performing a competitionexperiment. Specifically, when the binding of the anti-EP4 antibody toEP4 described in Examples is competitively inhibited by a secondaryanti-EP4 antibody, it is determined that the primary antibody and thesecondary antibody bind to substantially the same antigenic site. Thus,an antibody binding to substantially the same site as the EP4 bindingsite of the antibody isolated in Examples, which has an action toinhibit the function of EP4, is included in the present invention.

Method for Obtaining the Antibody of the Present Invention

The anti-EP4 antibody of the present invention may be a monoclonalantibody, a polyclonal antibody, or a functional fragment thereof. Ofthese, a monoclonal antibody is preferable because it can stably producean antibody that is homogenous as a pharmaceutical composition. The term“monoclonal” suggests the properties of an antibody obtained from agroup of substantially homogenous antibodies. Thus, this term is notused to mean that the antibody is produced by a specific method. Forinstance, the monoclonal antibody used in the present invention may beproduced, for example, by a hybridoma method (Kohler and Milstein,Nature 256: 495 (1975)) or a recombination method (U.S. Pat. No.4,816,567). The monoclonal antibody used in the present invention mayalso be isolated from a phage antibody library (Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1991)).Particular examples of the monoclonal antibody used in the presentinvention include: a “chimeric” antibody (immunoglobulin), in which aportion of the heavy chain and/or light chain of the monoclonal antibodyused in the present invention is derived from specific species or aspecific antibody class or subclass, and a remaining portion of thechain(s) is derived from another antibody class or subclass; an antibodyvariant: and a functional fragment thereof (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)).

When the antibody of the present invention is a polyclonal antibody, itcan be prepared by injecting a mixture of an immunogen and an adjuvantinto, for example, a mammalian host animal. In general, an antigen usedas an immunogen and/or an adjuvant are injected into the subcutis of ahost animal several times. Examples of such an adjuvant include aComplete Freund's adjuvant and monophosphoryl lipid A-trehalosedicorynomycolate (MPL-TDM). After completion of the treatment with animmunogen, a desired antibody against EP4, which has been generated inblood, can be obtained using, as indicators, EP4-binding specificity andan action to suppress the function of EP4.

On the other hand, when the antibody of the present invention is amonoclonal antibody, it can be prepared, for example, by a hybridomamethod.

This method includes the following 4 steps of: (i) immunizing a hostanimal or host animal-derived cells with a human EP4 protein; (ii)recovering monoclonal antibody-secreting (or potentially secreting)lymphocytes; (iii) fusing the lymphocytes with immortalized cells; and(iv) selecting cells that secrete a desired monoclonal antibody. Amouse, a rat, a guinea pig, a hamster, or another suitable host animalis selected as an animal to be immunized, and then, an immunogen isinjected into the selected animal.

After completion of the immunization, lymphocytes obtained from the hostanimal are fused with an immortalized cell line using a fusion agentsuch as polyethylene glycol, so as to establish hybridoma cells. Asfusion cells, a rat or mouse myeloma cell line is used, for example.After completion of the cell fusion, the cells are allowed to grow in asuitable medium that contains one or more substrates that inhibit thegrowth or survival of unfused lymphocytes and immortalized cell line.According to an ordinary technique, parent cells that lack the enzyme,hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), areused. In this case, hypoxanthine, aminopterin and thymidine are added toa medium that inhibits the growth of the HGPRT-deficient cells andallows the growth of hybridomas (HAT medium). From the thus obtainedhybridomas, those producing desired antibodies can be selected, andthen, a monoclonal antibody of interest can be obtained from a medium inwhich the hybridomas grow according to an ordinary method.

The thus prepared hybridomas are cultured in vitro, or are cultured invivo in the ascites of a mouse, a rat, a guinea pig, a hamster, etc., sothat an antibody of interest can be prepared from a culture supernatantor ascites.

The nucleic acid of the present invention encodes the heavy chainvariable region or light chain variable region of the antibody of thepresent invention. The nucleic acid of the present invention thatencodes the heavy chain variable region or light chain variable regionmay be inserted into a vector, and the vector may be then expressed incells.

The type of the vector is not particularly limited, and it may beselected, as appropriate, depending on the type of a host cell intowhich the vector is to be introduced, and the like. Also, the vector maybe introduced into host cells suitable for the expression of an antibody(e.g. mammalian cells such as CHO cells, yeast cells, insect cells,etc.), so that a recombinant antibody can be prepared.

Definition of the Chimeric Antibody of the Present Invention andProduction Method Thereof

The embodiment of the anti-EP4 antibody of the present inventionincludes a genetically recombinant antibody. The type of such agenetically recombinant antibody is not particularly limited. Examplesof the genetically recombinant antibody include a chimeric antibody, ahumanized antibody and a human antibody. The term “chimeric antibody” isused herein to mean an antibody in which an animal-derived variableregion is ligated to a different animal-derived constant region, andparticularly, an antibody in which a mouse-derived antibody variableregion is ligated to a human-derived antibody constant region (see Proc.Natl. Acad. Sci. U.S.A., 81, 6851-6855, (1984), etc.). When a chimericantibody is produced, an antibody comprising such a ligation of avariable region to a constant region can be easily constructed accordingto a genetic recombination technique well known to a person skilled inthe art. Herein, with regard to mouse-derived antibody variable regions,the heavy chain variable region preferably consists of the amino acidsequence shown in, for example, SEQ ID NO: 2 or SEQ ID NO: 12, and thelight chain variable region preferably consists of the amino acidsequence shown in, for example, SEQ ID NO: 4 or SEQ ID NO: 14. Thechimeric heavy chain or chimeric light chain of the present invention isinserted into a vector. The type of a vector is not particularlylimited. The vector may be selected, as appropriate, depending on thetype of a host cell into which it is to be introduced, and the like.Also, the vector may be introduced into host cells suitable for theexpression of an antibody (e.g. mammalian cells such as CHO cells, yeastcells, insect cells, etc.), so that a recombinant antibody can beprepared.

Definition of the Humanized Antibody of the Present Invention andProduction Method Thereof

The chimeric antibody of the present invention includes a human(ized)antibody. The humanized antibody is an antibody in which a frameworkregion is derived from a human and CDR is a mouse-derived region. Thehumanized antibody can be produced by first grafting CDR from thevariable region of a mouse antibody into a human variable region toreconstitute heavy chain and light chain variable regions, and thenligating the thus humanized, reconstituted human variable region to ahuman constant region. Such a method for producing a humanized antibodyis well known in the present technical field (see, for example, Nature,321, 522-525 (1986); J. Mol. Biol., 196, 901-917 (1987): Queen C et al.,Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989)). Herein, the type ofa mouse-derived CDR sequence used for the anti-EP4 antibody of thepresent invention is not limited. For instance, examples of the heavychain CDR1 to 3 include the amino acid sequences shown in SEQ ID NOS: 5to 7, and examples of the light chain CDR1 to 3 include the amino acidsequences shown in SEQ ID NOS: 8 to 10 and the amino acid sequencesshown in SEQ ID NOS: 18 to 20.

In order to allow a humanized antibody heavy chain or a humanizedantibody light chain to express in host cells, the humanized antibodyheavy chain or the humanized antibody light chain may be inserted into avector. The type of such a vector is not particularly limited, and itcan be selected, as appropriate, depending on the type of a host cellinto which it is to be introduced, and the like. Also, the vector may beintroduced into host cells suitable for the expression of an antibody(e.g. mammalian cells such as CHO cells, yeast cells, insect cells,etc.), and an antibody is reconstituted in the host cells, so that arecombinant antibody can be prepared.

Definition of the Human Antibody of the Present Invention and ProductionMethod Thereof

The human antibody (complete human antibody) is an antibody in which ahyper variable region that is an antigen-binding site of a variableregion, a remaining region of the variable region, and a constant regionhave the same structures as those of a human antibody. However, thehyper variable region may also be derived from another animal. Such ahuman antibody can be easily produced by a person skilled in the artaccording to a known technique. The human antibody can be obtained, forexample, by a method using a human antibody-producing mouse having ahuman chromosomal fragment comprising the heavy chain and light chaingenes of a human antibody (see Tomizuka, K. et al., Nature Genetics,(1997) 16, 133-143; Kuroiwa. Y. et. al., Nuc. Acids Res., (1998) 26,3447-3448; Yoshida, H. et. al., Animal Cell Technology: Basic andApplied Aspects, (1999) 10, 69-73 (Kitagawa, Y., Matsuda, T. and Iijima,S. eds.), Kluwer Academic Publishers: Tomizuka, K. et. al., Proc. Natl.Acad. Sci. USA, (2000) 97, 722-727, etc.), or by a method of obtaining aphage display-derived human antibody selected from a human antibodylibrary (Wormstone, I. M. et. al, Investigative Ophthalmology & VisualScience., (2002) 43 (7), 2301-8; Carmen, S. et. al., Briefings inFunctional Genomics and Proteomics, (2002) 1 (2), 189-203; Siriwardena,D. et. al., Opthalmology, (2002) 109 (3), 427-431, etc.).

Functional Fragment of the Antibody of the Present Invention

The functional fragment of the antibody of the present invention means apartial region of the anti-EP4 antibody. Examples of such a functionalfragment include Fab, Fab′, F(ab′)₂, Fv (a variable fragment ofantibody), a single-chain antibody (a heavy chain, a light chain, aheavy chain variable region, a light chain variable region, etc.), scFv,a diabody (a scFv dimer), dsFv (a disulfide-stabilized variable region),and a peptide comprising CDR as at least a portion thereof. Fab is anantibody fragment having antigen-binding activity obtained by digestingan antibody molecule with the protease papain, wherein about anN-terminal half of the heavy chain binds to the light chain as a wholevia a disulfide bond.

Fab can be produced by digesting an antibody molecule with papain toobtain a fragment thereof. Fab can also be produced by, for example,constituting a suitable expression vector into which DNA encoding theFab has been inserted, then introducing the vector into suitable hostcells (e.g. mammalian cells such as CHO cells, yeast cells, insectcells, etc.), and then allowing it to express in the cells.

F(ab′)₂ is an antibody fragment having antigen-binding activity obtainedby digesting an antibody molecule with the protease pepsin, which isslightly greater than Fab that binds to another Fab via a disulfide bondat hinge region. F(ab′)₂ can be obtained by digesting an antibodymolecule with the protease pepsin, or it can also be produced by bindingthe after-mentioned Fab to another Fab via a thioether bond or adisulfide bond. Alternatively, F(ab′)₂ can also be produced according toa genetic engineering method, as with Fab.

Fab′ is an antibody fragment having antigen-binding activity obtained bycleaving the disulfide bond at hinge region of the above-describedF(ab′)₂. Fab′ can also be produced according to a genetic engineeringmethod, as in the case of Fab and the like.

scFv is an antibody fragment having antigen-binding activity that is aVH-linker-VL or VL-linker-VH polypeptide, wherein a single heavy chainvariable region (VH) is ligated to a single light chain variable region(VL) using a suitable peptide linker. Such scFv can be produced byobtaining cDNAs encoding the heavy chain variable region and light chainvariable region of an antibody and then treating them according to agenetic engineering method.

Diabody is an antibody fragment having divalent antigen-bindingactivity, in which scFv is dimerized. Regarding the divalentantigen-binding activity, this activity may be an activity of binding toeither two identical antigens, or two different antigens. The diabodycan be produced by obtaining cDNAs encoding the heavy chain variableregion and light chain variable region of an antibody, then constructingcDNA expressing scFV, in which the heavy chain variable region isligated to the light chain variable region using a peptide linker, andthen treating the cDNA according to a genetic engineering method.

dsFv is a polypeptide which comprises a heavy chain variable region anda light chain variable region each including a substitution of one aminoacid residue with a cysteine residue, in which VH binds to VL via adisulfide bond between the cysteine residues. The amino acid residue tobe substituted with the cysteine residue can be selected based onantibody structure prediction according to the method of Reiter et al.or the like. Such dsFv can be produced by obtaining cDNAs encoding theheavy chain variable region and light chain variable region of theantibody and then constructing DNA encoding the dsFv according to agenetic engineering method.

A peptide comprising CDR is constituted such that it comprises at leastone region of CDR (CDR1-3) of the heavy chain or light chain. A peptide,which comprises multiple CDR regions, is able to bind to another peptidedirectly or via a suitable peptide linker. In the case of a peptidecomprising CDR, DNA encoding the CDR of the heavy chain or light chainof the antibody is constructed, and it is then inserted into anexpression vector. The type of the vector is not particularly limited,and it may be selected, as appropriate, depending on the type of a hostcells into which the vector is to be introduced, and the like. Thevector is introduced into host cells suitable for the expression of anantibody (e.g. mammalian cells such as CHO cells, yeast cells, insectcells, etc.), so that the peptide can be produced. Alternatively, such apeptide comprising CDR can also be produced by chemical synthesismethods such as an Fmoc method (fluorenylmethyloxycarbonyl method) and atBoc method (t-butyloxycarbonyl method).

Purification of the Antibody of the Present Invention

A method for purifying the antibody of the present invention is notparticularly limited, and a known method can be adopted. For example, aculture supernatant of the above-described hybridoma cells orrecombinant cells is recovered, and then, the antibody of the presentinvention can be purified from the culture supernatant by the combineduse of known methods such as various types of chromatography,salting-out, dialysis and membrane separation. When the isotype of anantibody is IgG, the antibody can be simply purified by affinitychromatography using protein A.

Medicament Comprising the Antibody of the Present Invention

The antibody of the present invention or a functional fragment thereofcan be used in a medicament comprising, as an active ingredient, theantibody or a functional fragment thereof. The medicament of the presentinvention can be used to treat or prevent EP4-related immunologicaldiseases, tumor, and pain.

Examples of the EP4-related immunological diseases include: psoriasis;multiple sclerosis; rheumatoid arthritis; systemic lupus erythematosus;inflammatory bowel diseases such as Crohn's disease; type I diabetes andcomplications thereof (e.g. diabetic retinopathy, diabeticmicroangiopathy, diabetic nephropathy, macular degeneration, etc.);polymyositis; Sjogren's syndrome; asthma; atopic dermatitis and contactdermatitis; immunodeficiency disorder; and organ transplantation.

The medicament of the present invention can be used to treat or preventpain, namely, nociceptive pain and neuropathic pain. Examples of thenociceptive pain include: pain caused by activation of somatic andvisceral nociceptors, such as pathologic deformation of articulationsand chronic arthralgia (e.g. arthritis including rheumatoid arthritis,osteoarthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis,and juvenile arthritis) (including the alleviation of disease and themaintenance of articular structure); lumbago and neck pain;musculoskeletal pain; myositis; bone fracture; distortion, bruise; painattended with fibromyalgia syndrome; pain associated with tumor and thetreatment thereof; pain attended with influenza or other viralinfectious diseases (common cold, etc.); rheumatic fever; visceral pain;pain attended with functional intestinal diseases (e.g. irritable bowelsyndrome, non-cardiac chest pain, non-ulcer dyspepsia, etc.); painattended with myocardial ischemia; dental pain; post-surgical andpost-dental-treatment pain; postpartum pain; primary headache disorder(e.g. migraine headache, tension headache, cluster headache, and otherprimary headache disorders); secondary headache disorder (e.g. headachecaused by the head and neck injury, headache caused by vascular disorderof the head and neck, headache caused by non-vascular intracranialdiseases, headache caused by substance abuse or substance withdrawal,headache caused by infectious diseases, headache caused by homeostaticdisorders, headache or prosopalgia caused by the disorders of thecranium, neck, eye, ear, nose, sinus, tooth, mouse or other face andcranium constitutional tissues, drug induced headache, and pain attendedwith migraine headache).

Examples of the neuropathic pain include: physical injury or ablation;phantom limb pain; pain caused by chronic inflammatory symptom:postherpetic neuralgia; diabetic neuropathy; nonspecific lumbago;backache; sciatica; neuropathy associated with tumor and the treatmentthereof; HIV-related neuropathy; carpal tunnel syndrome; chronicalcoholism; hypothyroidism; trigeminal neuralgia; trigeminal/autonomicheadache; uraemia; avitaminosis; multiple sclerosis; fibromyalgiasyndrome; and pain attended with toxin.

The medicament of the present invention is effective for the treatmentor prevention of tumor. The meaning of the treatment of tumor includesnot only the entire or partial inhibition of the growth, diffusion ormetastasis of tumor, or the entire or partial elimination of tumorcells, but it also includes the partial or entire resolution of symptomsattended with the tumor (pain, anorexia, weight reduction, etc.).

The treatment or prevention of tumor is directed at the overgrowth ofbenign tumor and polyp, the overgrowth of malignant tumor and polyp, andneoplasm.

Examples of the overgrowth of benign tumor and polyp include: squamouscell papilloma; basal cell carcinoma; transitional cell papilloma;adenocarcinoma; gastrinoma; cholangiocellular adenoma; hepatocellularadenoma; nephridial adenoma; oncocytoma; glomus tumor; melanocyticnevus; fibroma; myxoma; lipoma; leiomyoma: rhabdomyoma; benign teratoma;angioma; osteoma; chondroma; and meningioma.

Examples of the overgrowth of malignant tumor and polyp include: hepaticcell carcinoma; cholangiocarcinoma; renal cell carcinoma; squamous-cellcarcinoma: basal cell carcinoma; transitional cell carcinoma;adenocarcinoma; malignant gastrinoma; malignant melanoma; fibrosarcoma;myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; malignantteratoma; angiosarcoma; Kaposi's sarcoma; osteosarcoma; chondrosarcoma;lymphangiosarcoma: malignant meningioma: non Hodgkin's lymphoma:Hodgkin's lymphoma; leukemia; and encephaloma.

Examples of the neoplasm include: epithelial cell-derived neoplasm(epithelial carcinoma), basal cell carcinoma, and adenocarcinoma; labialcancer, oral cancer, esophageal cancer, gastrointestinal cancer such assmall intestinal cancer and stomach cancer, colon cancer, rectal cancer,liver cancer, bladder cancer, pancreatic cancer, ovarian cancer,cervical cancer, lung cancer, and breast cancer; skin cancer such assquamous-cell carcinoma and basal cell carcinoma, prostatic cancer, andrenal cell carcinoma; and known other cancers that affect systemicepithelial, mesenchymal or blood cells.

There can be provided an antibody-drug conjugate which comprises theantibody of the present invention and a compound having antitumoractivity and/or cytotoxicity. According to a genetic recombinationtechnique, the protein toxin used as a compound having antitumoractivity and/or cytotoxicity is fused with an antibody gene on the gene,so that it can be expressed as a single protein. The thus obtainedprotein is generally referred to as immunotoxin. Examples of thecompound having antitumor activity include doxorubicin and mitomycin C.A method for producing an antibody-drug conjugate is not particularlylimited. An example of the method is a method of coupling an antibodywith a drug via a disulfide bond or a hydrazone bond.

Pharmaceutical Composition Comprising the Antibody of the PresentInvention

The present invention includes a medicament or a pharmaceuticalcomposition. In addition to the above-described antibody of the presentinvention or a functional fragment thereof, a physiologically acceptablesalt thereof may also be used as an active ingredient of the medicamentof the present invention. When an acidic group is present, examples ofsuch a salt include: alkaline metal and alkaline earth-metal salts suchas lithium, sodium, potassium, magnesium or calcium; amine salts such asammonia, methylamine, dimethylamine, trimethylamine, dicyclohexylamine,tris(hydroxymethyl)aminomethane, N,N-bis(hydroxyethyl)piperazine,2-amino-2-methyl-1-propanol, ethanolamine, N-methylglucamine orL-glucamine; and salts formed with basic amino acids such as lysine,δ-hydroxylysine or arginine. When a basic group is present, examples ofsuch a salt include: salts formed with mineral acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid orphosphoric acid; salts formed with organic acids such as methanesulfonicacid, benzenesulfonic acid, p-toluenesulfonic acid, acetic acid,propionic acid, tartaric acid, fumaric acid, maleic acid, malic acid,oxalic acid, succinic acid, citric acid, benzoic acid, mandelic acid,cinnamic acid, lactic acid, glycolic acid, glucuronic acid, ascorbicacid, nicotinic acid or salicylic acid; and salts formed with acidicamino acids such as aspartic acid or glutamic acid.

As the medicament of the present invention, the antibody of the presentinvention or a functional fragment thereof, which is an activeingredient of the medicament, may directly be administered. In general,however, the medicament of the present invention is desirablyadministered in the form of a pharmaceutical composition comprising oneor two or more pharmaceutical additives, as well as the antibody of thepresent invention or a functional fragment thereof used as an activeingredient. As an active ingredient of the medicament of the presentinvention, a combination of two or more types of the antibodies of thepresent invention or functional fragments thereof may be used. Knownother agents may also be added to the above-mentioned pharmaceuticalcomposition.

The type of the pharmaceutical composition is not particularly limited.Examples of the dosage form include a tablet, a capsule, a granule, apowder medicine, syrup, a suspension, a suppository, an ointment, acream, a gel, a patch, an inhalant, and an injection. Thesepharmaceutical preparations are prepared according to an ordinarymethod. In the case of a liquid agent, it may adopt a form in which theagent is dissolved or suspended in water or another suitable mediumbefore use. Moreover, in the case of a tablet or a granule, it may becoated according to a well-known method. In the case of an injection, itis prepared by dissolving the compound of the present invention inwater. The present compound may also be dissolved in a normal saline ora dextrose solution, as necessary, and a buffer or a preservative mayalso be added to such a solution. The pharmaceutical composition may beprovided in any given pharmaceutical form used for oral or parenteraladministration. For example, the pharmaceutical composition may beprepared in the form of a pharmaceutical composition used for oraladministration, such as a granule, a parvule, a powder medicine, a hardcapsule, a soft capsule, syrup, an emulsion, a suspension or a liquidagent, or it may be prepared in the form of a pharmaceutical compositionused for parenteral administration (intravenous administration,intramuscular administration or subcutaneous administration), such as aninjection, a drop, a percutaneous absorption agent, a transmucosalabsorption agent, a transnasal agent, an inhalant or a suppository. Inthe case of an injection or a drop, it may be prepared in a powdery formsuch as a freeze-dried form, and it may be then dissolved in a suitableaqueous medium such as a normal saline before use. Also, asustained-release preparation coated with a polymer or the like can bedirectly administered into the brain.

The types of pharmaceutical additives used in the production of thepharmaceutical composition, the ratio of the pharmaceutical additives tothe active ingredient, and a method for producing the pharmaceuticalcomposition may be appropriately determined by a person skilled in theart, depending on the form of the pharmaceutical composition to beproduced. As such pharmaceutical additives, inorganic or organicsubstances, or solid or liquid substances can be used. In general, suchpharmaceutical additives can be added at a weight percentage of 1% to90% based on the weight of the active ingredient. Specific examples ofsuch substances used as pharmaceutical additives include lactose,glucose, mannit, dextrin, cyclodextrin, starch, suclose, magnesiumaluminometasilicate, synthetic aluminum silicate, carboxymethylcellulosesodium, hydroxypropyl starch, carboxymethylcellulose calcium,ion-exchange resin, methyl cellulose, gelatin, gum Arabic, hydroxypropylcellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone,polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate,talc, tragacanth, bentonite, Veegum, titanium oxide, sorbitan fatty acidester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester,purified lanolin, glycerinated gelatin, polysorbate, macrogol, vegetableoil, wax, liquid paraffin, white petrolatum, fluorocarbon, nonionicsurfactant, propylene glycol, and water.

In order to produce a solid preparation used for oral administration,the active ingredient is mixed with an excipient ingredient such aslactose, starch, crystalline cellulose, calcium lactate or silicic acidanhydride to prepare a powder medicine. Otherwise, a binder such assaccharose, hydroxypropyl cellulose or polyvinylpyrrolidone, adisintegrator such as carboxymethyl cellulose or carboxymethylcellulosecalcium, or other additives are further added to the above obtainedmixture, as necessary, and the obtained mixture is then subjected to wetgranulation or dry granulation, so as to prepare a granule. In order toproduce a tablet, such a powder medicine or a granule may be subjectedto a tablet-making operation, directly or with addition of a lubricantsuch as magnesium stearate or talc. The prepared granule or tablet maybe coated with an enteric coating base such ashydroxypropylmethylcellulose phthalate or a methacrylic acid-methylmethacrylate polymer, so as to prepare an enteric coated drug.Otherwise, it may be coated with ethyl cellulose, carnauba wax orhydrogenated oil, so as to prepare a long-acting preparation. Moreover,in order to produce a capsule, a powder medicine or a granule is filledinto a hard capsule, or the active ingredient is coated with a gelatinfilm, directly or after being dissolved in glycerin, polyethyleneglycol, sesame oil, olive oil or the like, thereby producing a softcapsule.

In order to produce an injection, the active ingredient, together with apH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lacticacid, sodium, sodium monohydrogen phosphate or sodium dihydrogenphosphate, and an isotonizing agent such as sodium chloride or glucose,as necessary, is dissolved in a distilled water for injection, and theobtained solution is subjected to aseptic filtration and is then filledinto an ampule. Otherwise, mannitol, dextrin, cyclodextrin, gelatin orthe like may be further added to the above obtained solution, and theobtained mixture may be then subjected to vacuum freeze-drying, so as toprepare an injection which is dissolved before use. Furthermore,lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil or thelike is added to the active ingredient, and it is emulsified in water,so as to prepare an emulsion for injection.

In order to produce a rectal administration agent, the activeingredient, together with a suppository base such as cacao butter, tri-,di- and mono-glyceride of fatty acid, or polyethylene glycol, ismoisturized and melted, and the resultant is then poured into a mold,followed by cooling. Otherwise, the active ingredient may be dissolvedin polyethylene glycol or soybean oil or the like, and it may be thencoated with a gelatin film or the like.

The dose and administration frequency of the medicament of the presentinvention are not particularly limited. These factors can beappropriately selected by a doctor's judgment, depending on conditionssuch as the purpose of preventing and/or treating deterioration and/orprogression of a disease to be treated, the type of the disease, thebody weight and age of a patient, and the severity of the disease. Ingeneral, the dose of the present medicament is approximately 0.01 to1000 mg (the weight of the active ingredient) per adult per day via oraladministration. The medicament can be applied once or divided overseveral administrations per day, or every several days. When themedicament is used as an injection, it is desirable that the medicamentbe continuously or intermittently administered at a dose of 0.001 to 400mg (the weight of the active ingredient) per adult per day.

The medicament of the present invention can be prepared as asustained-release preparation such as an implantable tablet and adelivery system encapsulated into a microcapsule, using a carriercapable of preventing the sustained-release preparation from immediatelybeing removed from the body. Examples of the carrier that can be usedherein include biodegradable and biocompatible polymers, such asethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen,polyorthoester, and polylactic acid. Such materials can be easilyprepared by a person skilled in the art. In addition, a liposomesuspension can also be used as a pharmaceutically acceptable carrier.The type of useful liposome is not limited. Such liposome is prepared asa lipid composition comprising phosphatidyl choline, cholesterol andPEG-induced phosphatidylethanol (PEG-PE) by being passed through afilter with a suitable pore size, such that it has a size suitable foruse, and it is then purified by a reverse phase evaporation method.

The medicament of the present invention can be prepared as apharmaceutical composition in the form of a kit, and it can be includedin a container or package, together with an instruction manual foradministration. When the pharmaceutical composition of the presentinvention is provided in the form of a kit, different constituents inthe composition are wrapped with different containers, and they are thenmixed immediately before use. Thus, different constituents are wrapped,separately, because it makes possible to preserve active constituentsfor a long period of time without losing the functions of the activeconstituents.

A reagent contained in the kit is supplied into a container made of amaterial that effectively keeps the activity of the constituents for along period of time, does not adsorb the constituents on the innersurface thereof, and does not alter the quality of the constituents. Forexample, a sealed glass ampule may comprise a buffer enclosed in thepresence of neutral non-reactive gas such as nitrogen gas. The ampule isconstituted with glass, an organic polymer such as polycarbonate orpolystyrene, ceramic, metal, or any other suitable materials that arecommonly used to retain the reagent.

Moreover, the kit may also comprise an instruction manual. Theinstruction manual for the present kit may be printed on a paper and/ormay be recorded on an electrically or electromagnetically readablemedium, such as a floppy (registered trademark) disk, CD-ROM, DVD-ROM, aZip disk, a videotape or an audiotape, and it may be then provided to auser in such a form. A detailed instruction manual may be actuallyincluded with the kit, or it may be published on the website that isdesignated by a kit manufacturer or distributor or noticed through ane-mail or the like.

Furthermore, the present invention includes a method for preventing ortreating EP4-related immunological diseases, tumor and pain, whichcomprises administering to a patient and the like the medicament orpharmaceutical composition of the present invention.

The term “treat” is used herein to mean inhibition or alleviation of theprogression and deterioration of the pathological condition of a mammalaffected with a disease that develops due to abnormality in the functionof EP4 (e.g. abnormal increase in the function, etc.). Thus, this is atreatment carried out for the purpose of inhibiting or alleviating theprogression and deterioration of the above-mentioned disease.

On the other hand, the term “prevent” is used herein to mean previousinhibition of the development of, or affecting with a disease thatdevelops due to abnormality in the function of EP4 (e.g. abnormalincrease in the function, etc.) in a mammal that is likely to beaffected with the aforementioned disease. Thus, this is a treatmentcarried out for the purpose of previously inhibiting the development ofvarious symptoms of the disease.

The “mammal” to be treated means any given animal belonging to Mammalia,and the type of the mammal is not particularly limited. Examples of themammal used herein include humans, pet animals such as a dog, a cat or arabbit, and livestock animals such as a bovine, a swine, sheep or ahorse. The particularly preferred “mammal” is a human.

The Antibody-Immobilized Carrier of the Present Invention

The present invention includes an antibody-immobilized carrier. Theantibody-immobilized carrier of the present invention is formed byimmobilizing the anti-human EP4 antibody of the present invention on acarrier. In a preferred embodiment, the antibody-immobilized carrier ofthe present invention is allowed to come into contact with bloodcontaining EP4-expressing cells, so that it can be used to remove theEP4-expressing cells from the body fluid. The anti-human EP4 antibodyimmobilized on a carrier may be of only one type, or of two or moretypes.

A specific form of the antibody-immobilized carrier of the presentinvention is, for example, the antibody of the present inventionimmobilized on a water-insoluble carrier, which is then filled into acontainer. Herein, all types of materials can be used as suchwater-insoluble carriers. In terms of moldability, sterilization and lowcytotoxicity, preferred materials include: synthetic polymers such aspolyethylene, polypropylene, polystyrene, acrylic resin, nylon,polyester, polycarbonate, polyacrylamide or polyurethane; naturalpolymers such as agarose, cellulose, cellulose acetate, chitin, chitosanor alginate; inorganic materials such as hydroxyapatite, glass, aluminaor titania; and metallic materials such as stainless steel or titanium.

Examples of the form of a carrier include a granular form, a flocculentform, a woven fabric, a non-woven fabric, a spongy porous form, and aplaty form. From the viewpoint of a large surface area per volume, agranular form, a flocculent form, a woven fabric, a non-woven fabric,and a spongy porous form are preferable. For example, peripheral bloodis supplied through a porous filter in which a container has previouslybeen filled with an antibody-immobilized water-insoluble carrier, sothat disease-associated EP4-expressing cells can be efficiently removed.

The antibody-immobilized carrier of the present invention can becombined with other components, so as to produce a kit for removingEP4-expressing cells. Examples of other components include ananticoagulant and an extracorporeal circulation circuit.

Diagnostic Kit Comprising the Anti-EP4 Antibody of the Present Invention

The anti-EP4 antibody of the present invention can be provided in theform of a diagnostic kit. The diagnostic kit of the present inventioncomprises an antibody, and may also comprise a labeling substance, or asecondary antibody or a labeled substance thereof. The labeled substanceof antibody means an antibody labeled with an enzyme, a radioisotope, afluorescent compound, a chemiluminescent compound, etc. In addition tothe aforementioned components, the diagnostic kit of the presentinvention may comprise other reagents used for carrying out thedetection of the present invention, for example, if the labeledsubstance is an enzyme-labeled substance, the diagnostic kit may alsocomprise an enzyme substrate (a coloring substrate, etc.), an enzymesubstrate solution, an enzyme reaction termination solution, an analytediluent, and the like. Furthermore, the present diagnostic kit may alsocomprise various types of buffers, sterilized water, various types ofcell culture vessels, various types of reactors (e.g. Eppendorf tube,etc.), a blocking agent (Bovine Serum Albumin (BSA), Skim milk, andserum components such as Goat serum), a washing agent, a surfactant,various types of plates, an antiseptic agent such as sodium azide, anexperimental operation manual (instruction manual), and the like.Examples of the measurement method applied herein include ELISA. EI,RIA, fluorescence immunoassay (FIA), luminescence immunoassay, and flowcytometry. Among these methods, flow cytometry is particularlypreferable in terms of simplicity and high sensitivity. In addition, thediagnostic kit of the present invention can be used in combination withanother antibody kit comprising an antibody that recognizes a cellsurface antigen.

The diagnostic kit of the present invention is allowed to react with theblood cells of a patient affected with cancer, autoimmune disease or thelike, so that the ratio of EP4-expressing cells in the blood can bedetected. By combining the present diagnostic kit with another cellsurface antigen antibody, the ratio of EP4-expressing cells in aspecific cell population (e.g. dendritic cells, TH17 cells, or Tregcells) can be detected. By evaluating an increase or decrease in theratio of the EP4-expressing cells, the condition of the disease can beevaluated.

Hereinafter, the present invention will be described more in detail inthe following examples. However, these examples are not intended tolimit the scope of the present invention.

EXAMPLES (1) Production of Human EP4 Expression Vector pcDNA-DEST40-hEP4

The sequence 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCC AGGTTCCACTGGTGAC-3′ (SEQID NO: 30) was added to the 5′-terminus of a sequence, which had beenprepared by removing a stop codon from the ORF sequence of the human EP4gene registered in Genbank (Accession No. NM_(—)000958), and thesequence 5′-GACCCAGCTTTCTTGTACAAAGTGGTCCCC-3′ (SEQ ID NO: 31) was addedto the 3′-terminus thereof, thereby synthesizing DNA. Using GatewaySystem (Invitrogen), the thus synthesized DNA was incorporated into apDONR221 vector (manufactured by Invitrogen), so as to preparepDONR-hEP4. The nucleotide sequence of the insert was determinedaccording to an ordinary method, and it was confirmed that the sequenceincluded no errors. Subsequently, using Gateway System, the sequencecontaining the human EP4 gene was incorporated into the pcDNA-DEST40vector (manufactured by Invitrogen) to obtain pcDNA-DEST40-hEP4. HumanEP4 expressed from this plasmid is a fusion protein in which V5 and6×HIS tags were added to the C-terminus. The plasmid DNA ofpcDNA-DEST40-hEP4 was prepared by transforming Escherichia coli (DH5α)according to an ordinary method and then amplifying it, and then usingPureLink HiPure Plasmid Filter Maxiprep Kit (manufactured by Invitrogen)in accordance with an instruction manual included therewith.

(2) Preparation of Human EP4-Expressing 293FT Cells

10 μg of the above-described plasmid DNA of pcDNA-DEST40-hEP4 wasintroduced into 293FT cells (manufactured by Invitrogen) plated on a100-mm collagen I coated cell culture dish, using 25 μL of Lipofectamin2000 (manufactured by Invitrogen) in accordance with an instructionmanual included therewith. Twenty-four hours after the geneintroduction, the cells were washed with HBSS (Hanks' Balanced SaltSolutions, manufactured by Invitrogen), were then removed from the cellculture dish using an enzyme-free cell dissociation buffer (manufacturedby Invitrogen), and were then recovered by centrifugation. The EP4gene-introduced 293FT cells and the gene-non-introduced 293FT cells weresubjected to a cell membrane permeabilization using Cytofix/Cytoperm Kit(manufactured by BD). The resulting cells were mixed with an anti-V5 tagantibody (manufactured by Invitrogen), and the mixture was thenincubated (4° C., 1 hour). Thereafter, the resultant was washed with awashing buffer (0.1% fetal bovine serum-containing PBS (PhosphateBuffered Saline, manufactured by Invitrogen)) three times, and was thenstained with an Alexa488-labeled anti-mouse IgG antibody (manufacturedby Invitrogen) used as a secondary antibody (4° C., 1 hour). Then, theresultant was again washed with a washing buffer three times, and wasthen analyzed with the flow cytometer Quanta SC MPL (manufactured byBECKMAN COULTER). As a result, since Alexa488 fluorescence positivesignal was detected in only the gene-introduced 293FT cells, it could beconfirmed that human EP4 was expressed in the cells.

Thus, this human EP4-expressing 293FT cell was used as a sensitizingantigen.

(3) Immunization

Antigen immunization was performed on 7-week-old female 129/Olabackground mice. The human EP4-expressing 293FT cells described in (2)above were suspended in a normal saline, and thereafter, the suspensionwere intraperitoneally administered to the above-mentioned mice 5 timesat administration intervals of 10 to 14 days.

(4) Production of Hybridoma

Three days after the 5^(th) immunization, the spleen was removed fromeach mouse, and splenic cells were prepared. The splenic cells and mousemyeloma P3X63Ag8.653 cells (obtained from ECACC) were subjected to cellfusion according to an ordinary method using Polyethylene Glycol 4000(manufactured by Merck). The fused cells were suspended in GIT Medium(manufactured by Wako Pure Chemical Industries, Ltd.) that contained 100units/mL penicillin, 100 μg/mL streptomycin, non-essential amino acid, 2mM L-glutamine, and NCTC-109 medium (all of which were manufactured byInvitrogen). The obtained suspension was then plated at a density of 100μL/well on a 96-well plate, and it was then cultured at 37° C. in 5%CO₂. From the day following the cell fusion, the medium was exchangedwith a medium formed by adding HAT Supplement (manufactured byInvitrogen) to the above-mentioned medium, and the culture was continuedfor 13 days after completion of the cell fusion. As a result, a colonyof hybridomas (approximately 700 clones) was obtained.

(5) Construction of NS0 Cell Line Stably Expressing Human EP4

Using Gateway System, the recombination reaction between the pDONR-hEP4described in (1) above and the pEF-DEST51 vector (manufactured byInvitrogen) was performed to obtain a plasmid pEF-DEST51-hEP4. Human EP4expressed by this plasmid is a fusion protein in which V5 and 6×HIS tagswere added to the C-terminus.

Using 35 μL of Lipofectamin LTX (manufactured by Invitrogen) and 14 μLof PlusReagent (manufactured by Invitrogen) in accordance with aninstruction manual included therewith, 14 μg of the plasmidpEF-DEST51-hEP4 was introduced into 1×10⁷ mouse myeloma NS0 cells(obtained from Cell Bank, RIKEN BioResource Center) that had beencultured in RPMI medium containing 10% fetal bovine serum, 100 units/mLpenicillin, and 100 μg/mL streptomycin (manufactured by Invitrogen).From the day following the gene introduction, while the medium wasexchanged with RPMI medium supplemented with 2.5 μg/mL antibioticblasticidin (manufactured by Invitrogen) every 3 days, the culture wascontinuously carried out for 2 weeks. Blasticidin-resistant NS0 cellswere cloned from the formed colonies according to a penicillin cupmethod.

The obtained blasticidin-resistant NS0 cells were blocked with FcBlock(manufactured by Becton, Dickinson and Company) at 4° C. for 15 minutes,and thereafter, the expression of an EP4 fusion protein was confirmedwith the flow cytometer by the same method as described in (2) above. Asa result, it could be confirmed that the obtained blasticidin-resistantNS0 cells stably expressed human EP4.

(6) Screening for Anti-EP4 Antibody-Producing Hybridoma

The NS0 cell line stably expressing human EP4 (2×10⁵ cells) produced in(5) above was stained by the same method as described in (2) above, andwas then analyzed with a flow cytometer. A cell membranepermeabilization was not performed, and 50 μL of culture supernatant ofthe hybridoma obtained in (4) above was used as a primary antibody. As aresult, an Alexa488 fluorescence positive reaction was found insupernatants in 21 wells. The cells in the positive well were cloned bylimiting dilution. A culture supernatant after completion of the culturefor 2 weeks was also subjected to a binding test with the NS0 cell linestably expressing human EP4 with a flow cytometer. The same cloning andbinding test were repeated again, and 2 clones of anti-EP4antibody-producing hybridomas were finally obtained. These hybridomaswere named as NBG016-mAb14 and NBG016-mAb21.

The obtained hybridoma cells NBG0116-mAb14 and NBG016-mAb21 weredeposited with the International Patent Organism Depositary, theNational Institute of Advanced Industrial Science and Technology, anIndependent Administrative Institution under the Ministry of Economy,Trade and Industry, at the AIST Tsukuba Central 6, Higashi 1-1-1,Tsukuba, Ibaraki, Japan (postal code: 305-8566), under accession Nos.FERM P-21978 and FERM P-21979, respectively, on Jun. 29, 2010 (theoriginal deposition date). Thereafter, the original deposition was thentransferred to an international deposition under the provisions of theBudapest Treaty (the notification date of “Certificate of Receipt ofOriginal Deposition” and “Viability Certification”: Sep. 5, 2011).Accession Nos. are FERM BP-11402 and FERM BP-11403, respectively.

(7) Purification of Anti-EP4 Antibody

The hybridoma cells NBG016-mAb14 and NBG016-mAb21 were each continuouslycultured in a serum-free CD-Hybridoma Medium (manufactured byInvitrogen), until approximately 90% of cells died, so as to produceantibodies. The cells were removed from 100 mL of the culturesupernatant by centrifugation (1,500 rpm, 15 minutes), and the residuewas then passed through HiTrap Protein G HP Column (manufactured by GEHealthcare Japan) to purify and concentrate IgG. To determine thesubtype of an IgG and the type of a light chain, the thus purified IgGwas examined using Iso Strip mouse monoclonal antibody isotyping kit(manufactured by Roche Diagnostics). As a result, they were both (IgG2a,κ). Hereinafter, the terms “NBG016-mAb14” and “NBG016-mAb21” indicatethese purified antibodies. When the term “hybridoma” or “cell” is used,such term indicates a hybridoma that produces these antibodies.

As in the case of (2), (3), (4) and (6) above, hybridoma cells producingan anti-EP4 antibody with a different subtype were obtained. From aculture supernatant of this hybridoma, purified IgG, the subtype andlight chain type of which were (IgG1, κ), was obtained in the samemanner as described in (7) above. This purified antibody was referred toas NBG016-mAb9.

(8) Production of the CHO Cell Line Stably Expressing Human EP4

Using Gateway System, a human EP4 gene from pDONR-hEP4 was incorporatedinto the pEF5/FRT/V5-DEST vector (manufactured by Invitrogen) to obtainpEF-FRT-hEP4. Human EP4 expressed by this plasmid is a fusion protein inwhich V5 and 6×HIS tags were added to the C-terminus.

Using Lipofectamin 2000, the plasmids pEF-FRT-hEP4 and pOG44(manufactured by Invitrogen) were simultaneously introduced intoFlp-In-CHO cells (manufactured by Invitrogen) that had been cultured inHam's F-12 medium (manufactured by Invitrogen) containing 10% fetalbovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin. Fromthe day following the gene introduction, the medium was exchanged withHam's F-12 medium supplemented with 500 μg/mL antibiotic hygromycin(manufactured by Invitrogen), and while exchanging the medium with afresh one every 3 days, the cells were cultured for 2 weeks. From theformed colonies, hygromycin-resistant cells were cloned according to apenicillin cup method.

A phycoerythrin (PE)-labeled anti-mouse IgG antibody (BECKMAN COULTER)was used as a secondary antibody, and the binding of the obtainedhygromycin-resistant cells to an anti-V5 tag antibody was analyzed witha flow cytometer by the method described in (2) above. As a result, theobtained hygromycin-resistant Flp-In-CHO cells showed the positivesignal of PE, and thus, it could be confirmed that the cells stablyexpressed human EP4. Hereinafter, this cell is referred to as a CHO cellline stably expressing human EP4.

(9) Binding Test of Anti-Human EP4 Antibody and Human EP4-ExpressingCells

The binding test of an anti-human EP4 antibody and a CHO cell linestably expressing human EP4 was carried out with flow cytometry by themethod described in (6) above. The CHO cell line stably expressing humanEP4 or the parent Flp-In-CHO cell line (5×10⁵ cells) was used. As aprimary antibody, 1 μg of NBG016-mAb14, NBG016-mAb21, or a mouse isotypecontrol antibody (manufactured by BioRegend) was used. As a secondaryantibody, a PE-labeled anti-mouse IgG antibody was used.

The results are shown in FIG. 1. The parent Flp-In-CHO cell line isindicated with the histogram filled with grey color, whereas the cellsstably expressing human EP4 are indicated with the black solid line.Both NBG016-mAb14 and NBG016-mAb21 bind only to the CHO cell line stablyexpressing human EP4. Thus, the results demonstrated that theseantibodies specifically bind to human EP4.

Likewise, NBG016-mAb9 was also subjected to a binding test with the CHOcell line stably expressing human EP4. The results are shown in FIG. 2.The results demonstrated that NBG016-mAb9 also specifically binds tohuman EP4.

(10) Inhibition Test Regarding PGE₂-Induced cAMP Production byAntibodies

The CHO cell line stably expressing human EP4 or the parent Flp-In-CHOcell line was cultured in a medium containing 1 mM acetylsalicylic acidfor 18 hours, and was then recovered from the cell culture dish using acell dissociation buffer. The recovered cells were then dispensed intoCulturPlate-96 (manufactured by PerkinElmer) at a density of 2,500 cellsper well. NBG016-mAb14, NBG016-mAb21, or a mouse isotype controlantibody was added to each well to a concentration of 0.05 to 30 μg/mL,and it was then left at room temperature for 15 minutes. Subsequently,PGE₂ (manufactured by Cayman) was added to each well to a concentrationof 5×10⁻¹¹ M, and the obtained mixture was further left at roomtemperature for minutes. Using LANCE Ultra cAMP Kit (manufactured byPerkinElmer), a reaction was carried out in accordance with aninstruction manual included with the kit. Then, the level of cAMPproduced in the cells was measured using the plate reader ARVO 1420 HTS(manufactured by PerkinElmer).

The results are shown in FIG. 3. When the mouse isotype control antibodywas added, it did not provide a significant inhibitory effect onPGE₂-induced cAMP production level. In contrast, when NBG016-mAb14 orNBG016-mAb21 was added, an inhibitory effect on cAMP production wasobserved in an antibody concentration-dependent manner. Using the dataanalysis software OriginPro 8.1 (manufactured by OriginLab), analysiswas carried out with logistic function. As a result, the IC₅₀ value ofNBG016-mAb14 was found to be 0.15 μg/mL (approximately 1.0 nM), and theIC₅₀ value of NBG016-mAb21 was found to be 0.24 μg/mL (approximately 1.6nM). From these results, it was demonstrated that NBG016-mAb14 andNBG016-mAb21 were functional antibodies having antagonist activity onhuman EP4, and that these antibodies have receptor function-inhibitingactivity that is equivalent to or greater than that of existingsubstances (e.g. low-molecular-weight compounds) having antagonistactivity on human EP4.

Likewise, NBG016-mAb9 was also subjected to a test involving addition of1.5×10⁻¹⁰ M PGE₂. The results are shown in FIG. 4. As a result of theabove-described analysis, the IC₅₀ value of NBG016-mAb9 was found to be4.6 μg/mL (approximately 28.8 nM). These results demonstrate thatNBG016-mAb9 is also a functional antibody having antagonist activity onhuman EP4.

(11) Production of Expression Vectors for Human EP1-4 and Mouse EP1-4

To the 5′-terminus of a sequence, which had been formed by removing astop codon from the ORF sequence of each of human EP1 (GenBank AccessionNo. NM_(—)000955), human EP2 (GenBank Accession No. NM_(—)000956), humanEP3a1 (GenBank Accession No. X83857), mouse EP1 (GenBank Accession No.NM_(—)013641), mouse EP2 (GenBank Accession No. NM_(—)008964), mouse EP3(GenBank Accession No. NM_(—)011196) and mouse EP4 (GenBank AccessionNo. NM_(—)008965), the sequenceCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGG TTCCAGGTTCCACTGGTGAC(SEQ ID NO: 32) was added, so as to prepare a DNA fragment. This DNAfragment was amplified by PCR using KOD FX (manufactured Toyobo Co.,Ltd.) according to an ordinary method. The amplified DNA wasincorporated into the pENTR/D-TOPO vector (manufactured by Invitrogen),so as to prepare pENTR-hEP1, pENTR-hEP2, pENTR-hEP3, pENTR-mEP1,pENTR-mEP2, pENTR-mEP3, and pENTR-mEP4. The nucleotide sequence of eachinsert was determined according to an ordinary method, and it wasconfirmed that the sequences included no errors. Using these 7 types ofplasmids and the plasmid pDONR-hEP4 produced in (1) above, individualinserts were incorporated into the pcDNA-DEST47 vector (manufactured byInvitrogen) by Gateway System. As a result, the following plasmids wereobtained: pcDNA-DEST47-hEP1, pcDNA-DEST47-hEP2, pcDNA-DEST47-hEP3,pcDNA-DEST47-hEP4, pcDNA-DEST47-mEP1, pcDNA-DEST47-mEP2,pcDNA-DEST47-mEP3, and pcDNA-DEST47-mEP4. These plasmids express afusion protein in which Cycle 3 Green Fluorescent Protein (GFP) has beenadded to the C-terminus of each PGE: receptor.

(12) Binding Specificity Test of Anti-EP4 Antibody

The pcDNA-DEST47-hEP1, pcDNA-DEST47-hEP2, pcDNA-DEST47-hEP3,pcDNA-DEST47-hEP4, pcDNA-DEST47-mEP1, pcDNA-DEST47-mEP2,pcDNA-DEST47-mEP3, and pcDNA-DEST47-mEP4 produced in (11) above (10 μgeach) were each introduced into 293FT cells, using Lipofectamin 2000. Onthe following day, the cells were washed with HBSS, and were thenremoved from the cell culture dish using an enzyme-free celldissociation buffer. The cells were then recovered by centrifugation.The thus recovered cells are referred to as 293FT cells transientlyexpressing EP.

The binding test of the anti-EP4 antibody of the present invention andthe 293FT transiently expressing EP cells was carried out with a flowcytometer according to the method described in (6) above. 293FT cells(5×10⁵ cells) that transiently expressed each of 8 types of PGE₂receptor subtypes were used. As a primary antibody. 1 μg of the anti-EP4antibody NBG016-mAb14 or NBG016-mAb21, or a mouse isotype controlantibody (manufactured by BioRegend) was used. As a secondary antibody,a PE-labeled anti-mouse IgG antibody was used.

As an example, the results of NBG016-mAb14 are shown in FIG. 5. Cellsshowing the positive signal of GFP-derived fluorescence were present,and thus, it could be confirmed that each PGE₂ receptor was expressed on239FT cells. However, among the 8 types of cells, those showing positivesignal of PE fluorescence were only human EP4-expressing cells. The sameresults were obtained from NBG016-mAb21. Thus, it was found that theanti-EP4 antibody of the present invention has strong specificity tohuman EP4. It was demonstrated that the present anti-EP4 antibody hasPGE₂ receptor subtype-binding specificity that is higher than that ofexisting substances having antagonist activity on human EP4.

Likewise, the binding specificity of NBG016-mAb9 was examined. Theresults are shown in FIG. 6. Among the 8 types of cells each expressingany one of the 8 types of PGE₂ receptor subtypes, those showing positivesignal of PE fluorescence were only human EP4-expressing cells. Fromthese results, it was found that NBG016-mAb9 also has strong specificityto human EP4.

(13) Binding Test of Human Lymphocytes and Anti-EP4 Antibody

Frozen human peripheral blood mononuclear cells (manufactured byCellular Technology Ltd.) were thawed using CTL-Anti-Aggregate-WashSupplement (manufactured by Cellular Technology Ltd.) in accordance withan instruction manual included therewith.

The binding test of the anti-EP4 antibody of the present invention tohuman lymphocytes was carried out with a flow cytometer according to themethod described in (6) above. The prepared human peripheral bloodmononuclear cells (9×10⁵ cells) were mixed with 1.5 μg of NBG016-mAb14,NBG016-mAb21 or a mouse isotype control antibody, respectively as aprimary antibody, and then mixed with an Alexa488-labeled anti-mouse IgGantibody as a second antibody. When an analysis was carried out using aflow cytometer, a cell population is divided into a lymphocyte subsetand a monocyte/macrophage subset based on the dot plots offorward-scattered light and side scattered light, and the Alexa488fluorescence intensity of the lymphocyte subset was then examined.

The analysis results with a flow cytometer are shown in FIG. 7. Theresults of the mouse isotype control antibody are shown with thehistogram filled with grey color, whereas the results of the anti-EP4antibody are shown with the black solid line. Since a majority of thehuman lymphocyte subset showed a positive signal of Alexa488fluorescence only in the case of the reaction of the human lymphocyteswith the anti-EP4 antibody, it was found that the human lymphocytesbound to the anti-EP4 antibody. From these results, it became clear thatthe anti-EP4 antibody of the present invention has an ability to bind tohuman endogenous EP4.

Likewise, NBG016-mAb9 was also subjected to a binding confirmationexperiment with human lymphocytes. Human peripheral blood mononuclearcells were isolated from human fresh peripheral blood using Lymphoprep(manufactured by AXIS SHIELD) in accordance with an instruction manualincluded therewith, and thereafter, a CD14-netagive cellular fractionwas separated using Anti-Human CD14 Microbeads (manufactured by MiltenyiBiotec) and was used as a human lymphocyte subset. The subsequentbinding confirmation experiment was carried out as described above,using a fluorescein isothiocyanate (FITC)-labeled anti-mouse IgGantibody (manufactured by BECKMAN COULTER) as a secondary antibody. Asshown in FIG. 8, a majority of the human lymphocytes bound toNBG016-mAb9, and thus, it was found that NBG016-mAb9 could also bind tohuman endogenous EP4.

(14) Immunostaining of PMA-Stimulated THP1 with Anti-EP4 Antibody

The human monocytic THP1 cell line was plated on a 4-well culture slide(manufactured by Becton, Dickinson and Company) at a density of 1.5×10⁵cells per well with RPMI medium containing 100 nM PMA (Phorbol12-myristate 13-acetate, manufactured by Sigma-Aldrich), and it was thencultured for 3 days, so that the cells were differentiated intomacrophage-like cells. After the removal of the medium, the cells werewashed with PBS three times, and were then immobilized with 200 μL of 1%paraformaldehyde solution (wherein the cells were left at 4° C. for 30minutes). The cells were washed with PBS three times again. Thereafter,300 μL of 1% BSA (Bovine Serum Albumin, manufactured by Wako PureChemical Industries, Ltd.) that contained 1 mg/mL human gamma globulin(manufactured by Wako Pure Chemical Industries, Ltd.) was added to theresulting cells to block them (wherein the cells were left at rest atroom temperature for 20 minutes). Subsequently, the resulting cells werewashed with 0.1% Tween 20 (manufactured by MP Bio)-containing PBS(hereinafter referred to as a washing buffer for immunostaining) threetimes. Thereafter. 200 μL of NBG016-mAb14, NBG016-mAb21 or a mouseisotype control antibody, which had been adjusted to 1 mg/mL, was addedto the cells, and the obtained mixture was then incubated at 4° C. for 1hour. Thereafter, the resultant was washed with a washing buffer forimmunostaining three times, and it was then stained with an FITC-labeledanti-mouse IgG antibody used as a secondary antibody (4° C., 1 hour).The slide was washed with a washing buffer for immunostaining threetimes, and was finally sealed with Propidium Iodide (PI)-containingVECTASHIELD Mounting Medium (manufactured by Vector Laboratories). Theprepared slide was observed under a fluorescence microscope.

The fluorescence microscopic images of the immunostained THP1 cells areshown in FIG. 9. The left view is a stained image of the mouse isotypecontrol antibody, wherein only the cell nucleus (grey) stained with PIcan be observed in the center. The right view is a stained image of theanti-EP4 antibody, wherein FITC fluorescence (white) is observed in agranular state surrounding the cell nucleus. From these results, it wasfound that the antibody of the present invention binds to native EP4 onthe surface of the cell membrane of the microphage-like cells that havebeen differentiated from the THP1 cell line by PMA.

(15) Inhibition of Cytokine Suppressive Effect of PGE₂ by Anti-EP4Antibody

PGE₂ has been known to suppress, via EP4 or EP2, the production ofcytokine in microphage by LPS stimulation. Whether or not the antibodyof the present invention would be able to recover the suppression ofcytokine production by PGE₂ via EP4 was examined usingPMA-differentiated THP1 that expressed an EP4 receptor. The THP1 cellline was plated on a 48-well cell culture plate at a density of 2.5×10⁵cells per well with 100 nM PMA-containing RPMI medium. After completionof the culture for 3 days, the medium was exchanged with an RPMI mediumcontaining 3.0 μg/mL NBG016-mAb14, NBG016-mAb21 or a mouse isotypecontrol antibody, and the obtained mixture was then incubated for 30minutes. Subsequently, PGE₂ was added to the resultant at aconcentration of 20 nM, and the obtained mixture was further incubatedfor 30 minutes. Then, LPS was added to the resultant at a concentrationof 100 ng/mL, and the obtained mixture was further cultured for 18hours. Thereafter, a culture supernatant was recovered, and the amountof TNFα in the culture supernatant was measured using TNFα Human DuoSetKit (manufactured by R & D Systems) in accordance with an instructionmanual included therewith.

Meanwhile, 0.5 mL of AlamarBlue (manufactured by MorphoSys) diluted to10 times its volume with RPMI medium was added to the cells after therecovery of the culture supernatant. The obtained mixture was incubatedfor 4 hours, and thereafter, fluorescence intensity was measured usingPlate Reader ARVO 1420 HTS under conditions consisting of an excitationwavelength of 535 nm and a detection wavelength of 595 nm. Based on themeasurement results with this AlamarBlue, the ratio of relativesurviving cell counts among individual wells was obtained, and theamount of TNFα produced per surviving cell count ratio was calculated.The degree at which the antibody of the present invention would be ableto recover the suppression of cytokine production by PGE₂ was calculatedbased on the following standard. Specifically, the amount of TNFα in awell to which only LPS stimulation was given was defined as a recoverypercentage of 100%, and the amount of TNFα in a well to which LPSstimulation and PGE₂ were given was defined as a recovery percentage of0%. Under such conditions, the recovery percentage in a case in whichLPS stimulation, PGE₂ and the present antibody were given was obtained.

The test results are shown in Table 1. Even if the mouse isotype controlantibody was added, no significant change was found in the suppressionof TNFα production by PGE₂. However, it was found that when NBG016-mAb14or NBG016-mAb21 was added, TNFα production suppressed by PGE₂ wasrecovered up to approximately 50%. The same results were obtained fromtwo independent tests. From these results, it became clear thatNBG016-mAb14 and NBG016-mAb21 are functional antibodies havingantagonist activity on human endogenous EP4.

TABLE 1 Antibody TNFα level ± SD Recovery rate ± SD LPS PGE₂ (3.0 μg/mL)(pg/mL) (%) Test 1 − − − 177.1 + − − 550.0 ± 21.8 + + − 286.1 ± 16.7 + +NBG016-mAb14 423.4 ± 33.9 52.0 ± 12.9 + + NBG016-mAb21 469.1 ± 48.1 69.3± 18.2 + + Isotype control 275.9 ± 11.1 −3.9 ± 4.2   Test 2 − − − 68.2 ±8.6 + − − 403.5 ± 37.1 + + − 166.8 ± 15.7 + + NBG016-mAb14 281.5 ± 10.748.5 ± 4.5 + + NBG016-mAb21 286.1 ± 19.5 50.4 ± 8.3 + + Isotype control148.9 ± 15.4  −7.6 ± 6.5  

(16) Isolation and Analysis of cDNA Encoding Variable Region of Anti-EP4Antibody

Total RNA was extracted from approximately 1×10⁷ hybridoma cellsproducing anti-EP4 antibodies (NBG016-mAb14 and NBG016-mAb21) usingRneasy Mini Kit (manufactured by QIAGEN) in accordance with aninstruction manual included with the kit. PCR was carried out accordingto a 5′-RACE (rapid amplification of cDNA ends) method using a 5′/3′RACE kit, 2^(nd) Generation (manufactured by Roche Diagnostics), so asto amplify the variable region of a heavy chain or a light chain.Primers corresponding to mouse constant regions γ1 and κ were used as 3′primers. That is, the 3′ primers used for amplifying the heavy chainvariable region were 5′-AGGGGCCAGTGGATAGACCGATG-3′ (SEQ ID NO: 33) and5′-GGCTGTTGTTTTGGCTGCAGAGAC-3′ (SEQ ID NO: 34). On the other hand, the3′ primers used for amplifying the light chain variable region were5′-ACTGGATGGTGGGAAGATGGATAC-3′ (SEQ ID NO: 35) and5′-TGGATACAGTTGGTGCAGCATCAG-3′ (SEQ ID NO: 36). Subsequently, each ofthe obtained amplified fragments was electrophoresed on agarose gel, anda band was then excised. DNA was purified by melting the gel. Thepurified DNA was incorporated into T-Vector pMD20 (manufactured byTakara Bio Inc.). Thereafter, the nucleotide sequence was analyzed, andits amino acid sequence was then determined. The sequence reaction wascarried out using ABI Prism BigDye Terminator Cycle Sequencing ReadyReaction Kits Version 3.1 (Applied Biosystems), and the nucleotidesequence was determined using Applied Biosystems 3130×1 Genetic Analyzer(Applied Biosystems). As a result of the analysis of the nucleotidesequence, the nucleic acid sequence encoding the heavy chain variableregion of NBG016-mAb14 was as shown in SEQ ID NO: 1, and the nucleicacid sequence encoding the light chain variable region thereof was asshown in SEQ ID NO: 3. Moreover, the amino acid sequence of the heavychain variable region thereof was as shown in SEQ ID NO: 2, and theamino acid sequence of the light chain variable region thereof was asshown in SEQ ID NO: 4.

Furthermore, the nucleic acid sequence encoding the heavy chain variableregion of NBG016-mAb21 was as shown in SEQ ID NO: 11, and the nucleicacid sequence encoding the light chain variable region thereof was asshown in SEQ ID NO: 13. Moreover, the amino acid sequence of the heavychain variable region thereof was as shown in SEQ ID NO: 12, and theamino acid sequence of the light chain variable region thereof was asshown in SEQ ID NO: 14.

From the above-described results, the amino acid sequences of CDRregions defined by Kabat et al. ((1991) Sequences of Proteins ofImmunological Interest, Fifth edition, U.S. Department of Health andHuman Services, U.S. Government Printing Office) were clarified.

The heavy chain CDR1-3 sequences of NBG016-mAb14 were SEQ ID NOS: 5 to7, respectively. The light chain CDR1-3 sequences thereof were SEQ IDNOS: 8 to 10, respectively. In addition, the heavy chain CDR1-3sequences of NBG016-mAb21 were SEQ ID NOS: 15 to 17, respectively. Thelight chain CDR1-3 sequences thereof were SEQ ID NOS: 18 to 20,respectively. The heavy chain CDR sequences of both clones werecompletely identical to each other.

Regarding NBG016-mAb9 as well, according to the above-described method,the heavy chain variable region was amplified using the primers shown inSEQ ID NO: 33 and SEQ ID NO: 34, and the light chain variable region wasamplified using the primers shown in SEQ ID NO: 35 and SEQ ID NO: 36.Then, the sequences of variable regions were determined. The nucleicacid sequence encoding the heavy chain variable region of NBG016-mAb9was as shown in SEQ ID NO: 41, and the nucleic acid sequence encodingthe light chain variable region thereof was as shown in SEQ ID NO: 42.In addition, the amino acid sequence of the heavy chain variable regionwas as shown in SEQ ID NO: 43, and the amino acid sequence of the lightchain variable region thereof was as shown in SEQ ID NO: 44.

The heavy chain CDR1-3 sequences of NBG016-mAb9 were SEQ ID NOS: 45 to47, respectively. The light chain CDR1-3 sequences thereof were SEQ IDNOS: 48 to 50, respectively.

(17) Cloning of Anti-EP4 Antibody Gene

Using Oligo-dT Primer included with First Strand cDNA Synthesis Kit ForRT-PCR (AMV) (manufactured by Roche Diagnostics), cDNA was synthesizedin accordance with an instruction manual included therewith. Using thesynthesized cDNA as a template, the full-length heavy chain and lightchain genes of the anti-EP4 antibody of the present invention wereamplified by PCR. The 5′-terminal sides of the heavy chain and lightchain were designed using the nucleotide sequence determined by 5′-RACEas a reference, whereas the 3′-terminal sides thereof were designedusing a constant region-specific sequence as a reference. The 5′ primerused for amplifying the heavy chain gene was5′-CACTGACCCTACGCGTATGGAATGGAGATGGATCTTTCTCTTC-3′ (SEQ ID NO: 37), andthe 3′ primer therefor was5′-ATAAGAATGCGGCCGCTCATTTACCAGGAGAGTGGGAGAG-3′ (SEQ ID NO: 38). The 5′primers used for amplifying the light chain variable region were5′-TTGCAGCCAGGAACGCGTATGGACATGAGGACCCCTGCT-3′ (SEQ ID NO: 39) and5′-ATAAGAATGCGGCCGCTTAACACTCATTCCTGTTGAAGCT-3′ (SEQ ID NO: 40). Theobtained heavy chain and light chain amplification fragments werecleaved with the restriction enzymes MluI and NotI. Then, the heavychain was inserted into pEHX1.1 (manufactured by Toyobo Co., Ltd.), andthe light chain was inserted into the MluI-NotI site of pELX2.1(manufactured by Toyobo Co., Ltd.). Thereafter, their nucleotidesequences were analyzed, and the amino acid sequences thereof were thendetermined.

As a result of the analysis of the nucleotide sequences, the nucleicacid sequence encoding the heavy chain of NBG016-mAb14 was as shown inSEQ ID NO: 22, and the nucleic acid sequence encoding the light chainthereof was as shown in SEQ ID NO: 24. Moreover, the amino acid sequenceof the heavy chain thereof was as shown in SEQ ID NO: 23, and the aminoacid sequence of the light chain thereof was as shown in SEQ ID NO: 25.

Furthermore, the nucleic acid sequence encoding the heavy chain ofNBG016-mAb21 was as shown in SEQ ID NO: 26, and the nucleic acidsequence encoding the light chain thereof was as shown in SEQ ID NO: 28.Moreover, the amino acid sequence of the heavy chain thereof was asshown in SEQ ID NO: 27, and the amino acid sequence of the light chainthereof was as shown in SEQ ID NO: 29.

The amino acid sequences of the heavy chain and light chain variableregions were identical to the amino acid sequences analyzed by theabove-described 5′-RACE method.

Regarding NBG016-mAb9 as well, cDNA was synthesized according to theabove-described method, and using the synthesized cDNA as a template,the full-length heavy chain and light chain genes of NBG016-mAb9 wereamplified by PCR. The 5′ primer used for amplifying the heavy chain genewas 5′-CACTAGAGCCCCCATACGCGTATGGCTGTCCTGGTGCTGTTCC-3′ (SEQ ID NO: 51),and the 3′ primer therefor was5′-ATAAGAATGCGGCCGCTCATTTACCCGGAGAGTGGGAGAG-3′ (SEQ ID NO: 52). The 5′primers used for amplifying the light chain gene were5′-TCCTCAGGTTGCCTCACGCGTATGAAGTTGCCTGTTAG-3′ (SEQ ID NO: 53) and5′-ATAAGAATGCGGCCGCTTAACACTCATTCCTGTTGAAGCT-3′ (SEQ ID NO: 40). Theobtained heavy chain and light chain amplification fragments werecleaved with the restriction enzymes MluI and NotI. Then, the heavychain was inserted into pEHX1.1 (manufactured by Toyobo Co., Ltd.), andthe light chain was inserted into the MluI-NotI site of pELX2.1(manufactured by Toyobo Co., Ltd.). Thereafter, their nucleotidesequences were analyzed, and the amino acid sequences thereof were thendetermined.

The nucleic acid sequence encoding the heavy chain of NBG016-mAb9 was asshown in SEQ ID NO: 54, and the nucleic acid sequence encoding the lightchain thereof was as shown in SEQ ID NO: 55. In addition, the amino acidsequence of the heavy chain thereof was as shown in SEQ ID NO: 56, andthe amino acid sequence of the light chain thereof was as shown in SEQID NO: 57.

(18) Confirmation of Whether the Obtained Antibody Gene Sequences wouldEncode Anti-EP4 Antibody

Recombinant antibodies of NBG016-mAb14 and NBG016-mAb21 were producedusing Mammalian PowerExpress System (manufactured by Toyobo Co., Ltd.).That is, pELX2.1 into which the light chain gene had been inserted wascleaved with the restriction enzymes EcoRI and BglII, and was thenelectrophoresed on agarose gel to purify a fragment comprising the lightchain gene. The purified light chain gene fragment was inserted into theEcoRI-BglII site of pEHX1.1 into which the heavy chain gene had beeninserted, so as to produce a plasmid having the genes of both the lightchain and the heavy chain. This plasmid was introduced into 293FT cellsusing Lipofectamin 2000, so that the cells transiently expressed theantibody.

Seventy-two hours after completion of the transduction, a cell culturesupernatant was collected, and it was then subjected to a binding testwith the CHO cell line stably expressing human EP4 with flow cytometryaccording to the method described in (6) above. As a control, a culturesupernatant of antibody gene-non-introduced 293FT cells was used. As aresult, the recombinant antibodies NBG016-mAb14 and NBG016-mAb21secreted into such a culture supernatant maintained an ability to bindto human EP4. From these results, it could be confirmed that theantibody gene sequences obtained in (17) above encode the anti-EP4antibody.

A recombinant antibody of NBG016-mAb9 was also produced by theabove-described method. That is, pELX2.1 into which the light chain genehad been inserted was cleaved with the restriction enzymes SalI andSpeI, and was then electrophoresed on agarose gel to purify a fragmentcomprising the light chain gene. The purified light chain gene fragmentwas inserted into the SalI-SpeI site of pEHX1.1 into which the heavychain gene had been inserted, so as to produce a plasmid having thegenes of both the light chain and the heavy chain. This plasmid wasintroduced into 293FT cells, so that the cells transiently expressed theantibody.

Seventy-two hours after completion of the transduction, a cell culturesupernatant was collected, and it was then subjected to a binding testwith the CHO cell line stably expressing human EP4 with flow cytometryaccording to the method described in (6) above. As a control, a culturesupernatant of antibody gene-non-introduced 293FT cells was used. Theresults are shown in FIG. 10. In FIG. 10, the parent Flp-In-CHO cellline is indicated with the histogram filled with grey color, whereas theCHO cell line stably expressing human EP4 is indicated with the blacksolid line. Since the recombinant antibody NBG016-mAb9 secreted into theculture supernatant maintained an ability to bind to human EP4, it couldbe confirmed that the antibody gene sequences of NBG016-mAb9 obtained in(17) above encode the anti-EP4 antibody.

(19) Production of Recombinant Anti-EP4 Antibody Stably Expressing CHOCells

A vector comprising the light chain and heavy chain genes of each ofNBG016-mAb14 and NBG016-mAb21 produced in (18) above was cleaved withthe restriction enzyme SspI, and was then purified by ethanolprecipitation. Using Lipofectamin 2000, the resultant was transducedinto CHO-K1 cells (Cell Bank, RIKEN BioResource Center), and theobtained cells were then cultured in Ham's F12 medium containing 10%fetal bovine serum for 24 hours. Twenty-four hours later, theaforementioned medium was exchanged with another Ham's F12 mediumcontaining 10% fetal bovine serum and 10 μg/mL puromycin, and then, thecells were cultured for 12 days, while exchanging the medium with afresh one every 3 days. Twelve days later, a colony was separated by apenicillin cup method.

The separated CHO-K1 cells were plated on a 24-well plate, and were thencultured in Ham's F12 medium containing 10 μg/mL puromycin for 3 days.Three days later, the medium was exchanged with another Ham's F12 mediumcontaining 10 μg/mL puromycin (to which fetal bovine serum had not beenadded), and the culture was further carried out for 72 hours.Thereafter, a culture supernatant was recovered.

Mouse IgG in the culture supernatant was detected by ELISA. A series ofculture supernatant dilutions were prepared using PBS, were thendispensed into Maxisorp 96-well plate (manufactured by Nunc), and werethen left at 4° C. overnight. On the following day, 3% BSA (manufacturedby Sigma)-containing PBS was added to the culture, and the obtainedmixture was then left at room temperature for 1 hour for blocking. Theresultant was washed with 0.1% Tween 20-containing PBS, and a horseradish peroxidase (HRP)-labeled anti-mouse IgG antibody (manufactured byMillipore) diluted to 4,000 times with 1% BSA-containing PBS was thenadded to the resultant. The obtained mixture was left at roomtemperature for 1 hour. Thereafter, the reaction product was washed with0.1% Tween 20-containing PBS, and 100 μL of coloring reagent (SureblueTMB microwell peroxidase substrate, manufactured by Kirkegaard & PerryLaboratories) was then added thereto. The obtained mixture was left atroom temperature for 5 minutes, and 100 μL of 1 N sulfuric acid wasadded to the mixture to terminate the reaction. Then, the absorbance at450 nm was measured. As a result, the expression of IgG was confirmed ina culture supernatant of CHO-K1 cells established by introduction of avector comprising the light chain and heavy chain genes.

(20) Binding Test of Recombinant Anti-EP4 Antibodies and CHO Cell LineStably Expressing Human EP4

The recombinant antibody NBG016-mAb14 and the recombinant antibodyNBG016-mAb21 were purified from a culture supernatant of the cell lineestablished in (19) above in the same manner as in (7) above. A bindingtest of each of the obtained purified recombinant anti-EP4 antibodiesand the CHO cell line stably expressing human EP4 was carried out with aflow cytometer by the method described in (6) above. The purifiedrecombinant anti-EP4 antibody or mouse isotype control antibody was usedin an amount of 1 μg per 5×10⁵ cells. As a secondary antibody, aPE-labeled anti-mouse IgG antibody was used.

The results are shown in FIG. 11. The parent Flp-In-CHO cell line isindicated with the histogram filled with grey color, whereas the CHOcell line stably expressing EP4 is indicated with the black solid line.Both the recombinant antibody NBG016-mAb14 and the recombinant antibodyNBG016-mAb21 bound only to the CHO cell line stably expressing human EP4stably expressing. As a result, it was confirmed that the purifiedrecombinant anti-EP4 antibodies maintained an ability to bind to humanEP4.

(21) Expression Vector for Mouse IgG1 Antibody NBG016-mAb21

The subclass of the NBG016-mAb21 purified in (7) above was IgG2a. Thus,the subclass of the NBG016-mAb21 was modified to be IgG1 so as toproduce the mouse IgG1 antibody NBG016-mAb21. The mouse IgG1 antibodyNBG016-mAb21 gene was produced as follows by Overlapping PCR. Using theheavy chain gene of NBG016-mAb21 as a template, and also using5′-CACTGACCCTACGCGTATGGAATGGAGATGGATCTTTCTCTTC-3′ (SEQ ID NO: 37) and5′-GACAGATGGGGGTGTCGTTTTAGCGCTAGAGACAGTGACCAGAGTC CC-3′ (SEQ ID NO: 58),the variable region gene of NBG016-mAb21 was amplified by PCR. At thesame time, using cDNA synthesized from the total RNA of a hybridomaproducing mouse IgG1 as a template, and also using5′-GGGACTCTGGTCACTGTCTCTAGCGCTAAAACGACACCCCCATCTGT C-3′ (SEQ ID NO: 59)and 5′-ATAAGAATGCGGCCGCTCATTTACCAGGAGAGTGGGAGAG-3′ (SEQ ID NO: 38), amouse IgG1 portion ranging from CH1 to the constant region gene wasamplified by PCR. The thus amplified heavy chain variable region genewas mixed with the amplified CH1-constant region gene fragment, and theobtained mixture was then amplified by PCR using the primers of SEQ IDNO: 37 and SEQ ID NO: 38. The thus amplified DNA fragment was cleavedwith the restriction enzymes MluI and NotI, and the cleaved fragment wasthen inserted into the MluI-NotI site of the expression vector pEHX1.1.The obtained expression vector was cleaved with the restriction enzymesEcoRI and BglII, and a light chain gene fragment (EcoRI-BglII fragment)of NBG016-mAb21 was then inserted therein, so as to produce anexpression vector for the mouse IgG1 antibody NBG016-mAb2.

(22) Method for Producing Cell Line Stably Expressing Mouse IgG1Antibody NBG016-mAb21

Floating CHO-K1 cells (manufactured by Toyobo Co., Ltd.) (2.5×10⁵cells/ml) cultured in 8 mM glutamine-containing EX-CELL CD CHO medium(manufactured by SAFC Bioscience) were dispensed in an amount of 1 mlinto each of two wells of a 24-well plate. Thereafter, 136 μl ofOpti-MEM, 15 μl of Lipofectamin 2000, and 4 μg of expression vector formouse IgG1 antibody NBG016-mAb21 cleaved with the restriction enzymeSspI were blended, and the obtained mixture was left at room temperaturefor 20 minutes. Subsequently, the reaction product was added in anamount of 68 μl each to the wells containing CHO-K1, and the obtainedmixture was then incubated in a CO₂ incubator for 24 hours. Twenty-hourhours later, the cells were suspended in 8 ml of EX-CELL CD CHO mediumcontaining 8 mM glutamine, and the obtained suspension was dispensed inan amount of 4 ml into each of two wells of a 6-well plate. Then, 3 μlof 10 mg/ml puromycin was added to one well, and 4 μl of 10 mg/mlpuromycin was added to the other well. While exchanging the medium witha fresh one every 3 or 4 days, the cells were cultured for 18 days.Thereafter, proliferating cells were recovered from the well, and therecovered cells were then suspended in Conditioned medium (a mediumcontaining per ml: 700 ml of EX-CELL CD CHO medium, 300 ml of culturesupernatant of floating CHO-K1 cells, and 1 ml or 0.75 ml of 10 mg/mlpuromycin). The obtained suspension was dispensed in an amount of 200 μlinto each well of a 96-well plate. One week later, 100 μl of Conditionedmedium was added, and the obtained mixture was further culture for 1week. Thereafter, the cells were subcultured several times, and 500 μlof drug-resistant cells (4×10⁴ cells/ml) were then added to a 24-wellplate, followed by culture for 5 days. Five days later, the amount ofthe antibody in the culture supernatant was quantified using Mouse IgGEIA Kit (manufactured by Takara Bio Inc.), and antibody-producing cellswere then screened. The thus obtained cells were defined as a CHO cellline stably expressing mouse IgG1 antibody NBG016-mAb21.

(23) Purification of Mouse IgG1 Antibody NBG016-mAb21

The CHO cell line stably expressing mouse IgG1 antibody NBG016-mAb21 wascultured in EX-CELL CD CHO medium containing 8 mM glutamine and 7.5μg/ml puromycin for 10 days, so as to allow it to produce antibody. From200 mL of this culture supernatant, purified IgG was obtained in thesame manner as described in (7) above. Hereinafter, the obtainedpurified IgG was referred to as mouse IgG1 antibody NBG016-mAb21.

(24) Binding Test of Mouse IgG1 Antibody NBG016-mAb21 and Human EP4

A binding test of the mouse IgG1 antibody NBG016-mAb21 to the CHO cellline stably expressing human EP4 was carried out in the same manner asdescribed in (9) above. The results are shown in FIG. 12(A). The parentFlp-In-CHO cell line is indicated with the histogram filled with greycolor, whereas the CHO cell line stably expressing human EP4 isindicated with the black solid line. The mouse IgG1 antibodyNBG016-mAb21 bound only to the human EP4 stably expressing CHO cell linestably expressing human EP4. As a result, it was confirmed that theproduced mouse IgG1 antibody NBG016-mAb21 maintained an ability to bindto human EP4.

(25) Inhibitory Test Regarding PGE₂-Induced cAMP Production by MouseIgG1 Antibody NBG016-mAb21

Whether or not PGE₂-induced cAMP production would be inhibited by themouse IgG1 antibody NBG016-mAb21 was examined in the same manner asdescribed in (10) above. The cells were allowed to react with theantibody at room temperature for 15 minutes, and PGE₂ was then added tothe reaction mixture at a concentration of 1.5×10⁻¹⁰ M. The thusobtained mixture was further left at room temperature for 30 minutes.Using LANCE Ultra cAMP Kit (manufactured by PerkinElmer), and a reactionwas carried out in accordance with an instruction manual included withthe kit, so as to measure the level of cAMP.

The results are shown in FIG. 12(B). When a mouse isotype controlantibody was added, it did not provide a significant inhibitory effecton PGE₂-induced cAMP production level. In contrast, when the mouse IgG1antibody NBG016-mAb21 was added, a cAMP production inhibitory effect wasobserved in an antibody concentration-dependent manner. The IC₅₀ valuewas found to be 1.1 μg/mL (approximately 6.9 nM). From these results, itwas demonstrated that even if NBG016-mAb21 is modified to a mouse IgG1antibody, it could maintain antagonist activity on EP4.

(26) Production of Human Chimeric Antibodies NBG016-mAb14 andNBG016-mAb21

Using Mammalian PowerExpress System (manufactured by Toyobo Co., Ltd.),human chimeric antibodies in which the CH1 region and constant region ofNBG016-mAb14 or NBG016-mAb21 were substituted with those of a humanantibody gene. The heavy chain variable region gene of each ofNBG016-mAb14 and NBG016-mAb21 was amplified by PCR using5′-CACTGACCCTAAGCTTATGGAATGGAGATGGATCTTTCTCTTC-3′ (SEQ ID NO: 60) and5′-GGCTGTTGTGCTAGCTGCAGAGACAGTGACCAGAGT-3′ (SEQ ID NO: 61). The obtainedheavy chain gene fragment was cleaved with the restriction enzymesHindIII and NheI, and the cleaved fragment was then inserted into theHindIII-NheI site of the expression vector pEHγX1.1. At the same time,the light chain variable region gene of each of NBG016-mAb14 andNBG016-mAb21 was amplified by PCR using5′-ATTGCAGCCAGGAGAATTCATGGACATGAGGACCCCTGCT-3′ (SEQ ID NO: 62) and5′-GGTGCAGCATCCGTACGTTTTATTTCCAACTTTGTCCCC-3′ (SEQ ID NO: 63). Theobtained light chain gene fragment was cleaved with the restrictionenzymes BsiWI and EcoRI, and the cleaved fragment was then inserted intothe BsiWI-EcoRI site of the expression vector pELκX2.1. The light chaingene-inserted pELκ2.1 was cleaved with the restriction enzymes BglII,NotI and ScaI, and the cleaved fragment was then electrophoresed onagarose gel, so as to purify a fragment containing the light chain gene.The purified light chain gene fragment was incorporated into theBglII-NotI site of pEHγX1.1, into which the heavy chain gene had beeninserted, thereby producing a plasmid that maintained the genes of boththe light chain and the heavy chain. Using Lipofectamin 2000, thisplasmid was introduced into 293FT cells, so that the cells transientlyexpressed the antibody.

Seventy-two hours after completion of the introduction, a cell culturesupernatant was collected, and it was then subjected to a binding testwith the CHO cell line stably expressing human EP4 with flow cytometryaccording to the method described in (6) above. As a control, a culturesupernatant of antibody gene-non-introduced 293FT cells was used. As asecondary antibody, a PE-labeled anti-human IgG antibody (manufacturedby Abcam) was used. The results are shown in FIG. 13. In FIG. 13, theparent Flp-In-CHO cell line is indicated with the histogram filled withgrey color, whereas the CHO cell line stably expressing human EP4 isindicated with the black solid line. From the results shown in thefigure, it could be confirmed that the human chimeric antibodiesNBG016-mAb14 and NBG016-mAb21 secreted into the culture supernatant eachmaintained an ability to bind to human EP4.

These results demonstrated that the nucleic acid sequence encoding theantibody provided by the present invention can be used to produce arecombinant antibody (e.g. a chimeric antibody, a humanized antibody, ahuman antibody, etc.) that maintains the function of the antibody of thepresent invention.

INDUSTRIAL APPLICABILITY

Since the antibody provided by the present invention specificallysuppresses the function of a human PGE₂ receptor subtype EP4, thepresent antibody is anticipated to play an important role in providing amethod for preventing or treating EP4-related diseases or in thedevelopment of a preventive or therapeutic agent for the aforementioneddiseases.

[Sequence Listing]

TPC0033NBK-seq.ST25.txt

The invention claimed is:
 1. A monoclonal antibody, wherein the antibodyis produced from hybridomas with the international depositary accessionNos. FERM BP-11402 (NBG016-mAb14) and FERM BP-11403 (NBG016-mAb21).
 2. Amonoclonal antibody or an antigen-binding fragment thereof, comprising:heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:15, heavy chain CDR2 comprising the amino acid sequence shown in SEQ IDNO: 16, heavy chain CDR3 comprising the amino acid sequence shown in SEQID NO: 17, light chain CDR1 comprising the amino acid sequence shown inSEQ ID NO: 18, light chain CDR2 comprising the amino acid sequence shownin SEQ ID NO: 19, and light chain CDR3 comprising the amino acidsequence shown in SEQ ID NO: 20; wherein the antibody or antigen-bindingfragment specifically binds to the extracellular domain of EP4, inhibitsthe function of EP4, and binds to neither EP1, EP2 nor EP3.
 3. Themonoclonal antibody or antigen-binding fragment thereof according toclaim 2, wherein the antibody or antigen-binding fragment has: a heavychain variable region comprising the amino acid sequence shown in SEQ IDNO: 12, and a light chain variable region comprising the amino acidsequence shown in SEQ ID NO: 14; wherein the antibody or antigen-bindingfragment specifically binds to the extracellular domain of EP4, inhibitsthe function of EP4, and binds to neither EP1, EP2 nor EP3.
 4. Themonoclonal antibody or antigen-binding fragment thereof according toclaim 2, wherein the antibody is a humanized antibody or a chimericantibody.
 5. The monoclonal antibody or antigen-binding fragment thereofaccording to claim 2, wherein the antigen-binding fragment is Fab, Fab′,F(ab′)₂, Fv, scFv, a dimer of scFv or dsFv.
 6. A pharmaceuticalcomposition, comprising: the monoclonal antibody or antigen-bindingfragment thereof according to claim 2, or a physiologically acceptablesalt thereof, and a pharmaceutically acceptable additive.
 7. Anantibody-immobilized carrier, comprising: a carrier, and the monoclonalantibody or antigen-binding fragment thereof according to claim 2,wherein the antibody or antigen-binding fragment thereof is immobilizedon the carrier.
 8. A kit for measuring the expression level of EP4 on acell surface, comprising: the monoclonal antibody or antigen-bindingfragment thereof according to claim 2, and a container or package. 9.The monoclonal antibody or antigen-binding fragment thereof according toclaim 4, wherein the antigen-binding fragment is Fab, Fab′, F(ab′)₂, Fv,scFv, a dimer of scFv or dsFv.
 10. A pharmaceutical composition,comprising: the monoclonal antibody or antigen-binding fragment thereofaccording to claim 4, or a physiologically acceptable salt thereof, anda pharmaceutically acceptable additive.